Treatment of drug resistant gliomas

ABSTRACT

The present disclosure describes the use of 2,4-disulfonyl phenyl tert-butyl nitron (2,4-ds-PBN) in the treatment of temozolomide drug resistant gliomas. The 2,4-ds-PBN may be used combined with other chemo- and radiotherapies and surgery, including temozolomide, to reduce glioma occurrence, recurrence, spread, growth, metastasis, and vascularization, and to inhibit development of temozolomide resistance.

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 62/561,002, filed Sep. 20, 2017, the entirecontents of which are hereby incorporated by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing XML, which has beensubmitted electronically and is hereby incorporated by reference in itsentirety. Said Sequence Listing XML, created on Mar. 17, 2023, is namedOMRF.P0133US.xml and is −7kB in size.

BACKGROUND 1. Field

The present disclosure relates generally to the fields of oncology andchemotherapy. More particularly, it concerns the use of 2,4-disulfonylphenyl tert-butyl nitrone (2,4-ds-PBN) to treat drug resistant gliomas.

2. Description of Related Art

Gliomas are a diverse group of brain tumors that arise from normal“glial” cells of the brain and/or their precursor cells. The mostimportant determinant of survival for gliomas is the “grade” of theglioma. Secondary determinants of survival are age at diagnosis,performance status, and extent of surgery. Patients with low-gradegliomas have a protracted natural history with generally long survivaltimes, while those with high grade gliomas are much more difficult tosuccessfully treat and have shorter survival times. All gliomas havespecific signs and symptoms that are primarily related to the locationand size of the glioma.

The temporal lobe gliomas, for example, may cause seizures, difficultywith speech and/or loss of memory. The frontal lobe gliomas may causeseizures, behavioral changes, weakness of the arms or legs on theopposite side of the body, and/or difficulty with speech. The occipitalgliomas may cause loss of vision. The parietal gliomas may cause loss ofspatial orientation, diminished sensation on the opposite side of thebody, and/or inability to recognize once familiar objects or persons.

Astrocytomas are glioma tumors that arise from brain cells calledastrocytes or their precursors. Astrocytes are cells in the centralnervous system that support neuronal function. Astrocytomas can begraded by histologic features that signify increasing malignancy intoastrocytoma, anaplastic astrocytoma, or glioblastoma multiforme.Anaplastic astrocytoma and glioblastoma multiforme are consideredhigh-grade gliomas while the astrocytoma is considered to be a low-gradeglioma. High-grade tumors grow rapidly and can easily infiltrate andspread through the brain. Low-grade astrocytomas can also infiltrate thebrain but are usually more localized and grow slowly over a long periodof time. High-grade tumors are much more aggressive and require veryintense therapy. The majority of astrocytic tumors in children arelow-grade, whereas the majority in adults are high-grade. Astrocytomascan occur anywhere in the brain and spinal cord, however the majorityare located in the cerebral hemispheres.

Oligodendrogliomas are also gliomas. They arise from oligodendrocytesand/or their cell precursors. Normal oligodendrocytes provide myelin, afatty substance that covers nerve axons in the brain and spinal cord andallows nerves to conduct electrical impulses more efficiently.Oligodendrogliomas are classified as low grade oligodendroglioma (lessaggressive) and anaplastic oligodendroglioma (more aggressive). Morecommon than pure oligodendrogliomas are low grade and anaplastic tumorsthat are a mixture of astrocytoma and oligodendroglioma(“oligoastrocytomas”).

Anaplastic oligodendrogliomas and mixed oligoastrocytomas are moresensitive to cytotoxic chemotherapy than astrocytomas. A high rate ofresponse to the PCV (procarbazine (matulane), CCNU (lomustine),vincristine) chemotherapy has made the use of this regimen, if not thestandard of care for these tumors, at least a very common treatment. Lowgrade oligodendrogliomas are also sensitive to chemotherapy, and PCV canbe used when low grade tumors begin to grow despite priorsurgery/radiation therapy.

The efficiency of phenyl-tert-butyl-nitrone (PBN) as a potentialanti-glioma drug has been shown in the pre-treatment of a rat C6 gliomaimplantation model (Doblas et al., 2008). MRI results from untreatedrats showed the diffusive invasiveness of C6 gliomas, with someassociated angiogenesis. PBN administration as a pretreatment was foundto clearly induce a decrease in growth rate and tumor regression as wellas preventing angiogenesis. However, post-treatment of PBN had a reducedeffect (˜50% of tumors had reduced tumor growth) for tumor regressioncompared with pre-treatment (>80% of the tumors had reduced growth). MRIfindings rivaled those from histology and angiogenesis markerimmunostaining evaluations.

In a more recent study, the inventor has shown that a structural analogof PBN, 2,4-ds-PBN, decreased tumor volumes and delayed tumor growthrate. 2,4-ds-PBN post-treatment was also significantly effective inincreasing survival rate. This result was unexpected and surprising assulfonated derivatives of PBN were not known to be able to readily crossthe blood-brain-barrier (BBB). PBN, the parent nitrone, had beenpreviously found to easily penetrate the BBB (Wang & Shuaib, 2007).2,4-disulfonyl PBN (2,4-ds-PBN) is structurally related to the parentcompound PBN, but contains two sulfonyl groups that make it much morewater-soluble. As a result of the increased water solubility2,4-disulfonyl PBN was initially thought to not easily pass through theBBB, as compared to PBN (Wang & Shuaib, 2007). The inventor has howeverdemonstrated that 2,4-disulfonyl PBN can readily cross the BBB (Coutinhode Souza et al., 2015). Ideally, drugs that are to be used asanti-glioma therapy need to pass the endothelial junctions of the BBB toreach the majority of tumor cells (Cao et al., 2005), but it is possiblethat malignant gliomas have acquired the ability to actively degradetight junctions by secreting soluble factors, eventually leading to BBBdisruption within invaded brain tissue (Schneider et al., 2004).Specifically, 2,4-disulfonyl PBN has shown significant efficacy as amonotherapy for glioma tissue.

SUMMARY

Thus, in accordance with the present disclosure, there is provided amethod of treating a temozolomide-resistant glioma in a subjectcomprising administering to said subject a dose of 2,4-disulfonyl phenyltert-butyl nitrone (2,4-ds-PBN) effective to inhibit thevascularization, growth or spread of said glioma. Administration may bethrough a route requiring subsequent passage of 2,4-ds-PBN across theblood brain barrier, such as enteral, intravenous, or intra-arterial.

The subject may have a recurrent or metastatic glioma, and or may havepreviously failed one or more anti-glioma therapies. The effective doseof 2,4-ds-PBN may be from about 5 to about 150 mg/kg body weight perday. The enteral administration may be through dietary supplementationof a food component, or the enteral administration may be in the form ofa pill or a liquid. The subject may be a human.

The method may further comprise a secondary anti-glioma therapy, such asradiation, surgery, or chemotherapy, such as temozolomide, lomustine,vincristine, matulane, PCV,

BCNU, CCNU and/or DFMO. The secondary anti-glioma agent may betemozolomide, and the effective dose of temozolomide may be lower thanthe standard monotherapy dose of temozolomide.

The effective amount of 2,4-ds-PBN may be from about 0.005 w/w % toabout 0.1 w/w % of the diet being administered. The glioma may be anastrocytoma, an oligodendroglioma, or a glioblastoma multiforme, or aTGF-β1-, MGMT- and/or APGN-expressing form of any of the foregoing.

The method may further comprise assessing therapeutic efficacy bymeasuring the expression of liposaccharide binding protein prior to andafter a treatment with 2,4-ds-PBN. The method may further compriseassessing expression of LBP, wherein reduced LBP levels as compared tountreated controls indicates improved prognosis.

In another embodiment, there is provided a method for inhibiting ofdevelopment of glioma temozolomide resistance in a subject comprising(a) identifying a subject having a glioma and (b) administering to saidsubject doses of (i) 2,4-disulfonyl phenyl tert-butyl nitrone(2,4-ds-PBN), and (ii) temozolomide effective to inhibit the developmentof temozolomide resistance in said glioma. Administration may be througha route requiring subsequent passage of 2,4-ds-PBN across the bloodbrain barrier, such as enteral, intravenous, or intra-arterial.

The subject may have a recurrent or metastatic glioma, and or may havepreviously failed one or more anti-glioma therapies. The effective doseof 2,4-ds-PBN may be from about 5 to about 150 mg/kg body weight perday. The enteral administration may be through dietary supplementationof a food component, or the enteral administration may be in the form ofa pill or a liquid. The subject may be a human.

The method may further comprise a secondary anti-glioma therapy, such asradiation, surgery, or chemotherapy, such as temozolomide, lomustine,vincristine, matulane, PCV, BCNU, CCNU and/or DFMO. The secondaryanti-glioma agent may be temozolomide, and the effective dose oftemozolomide may be lower than the standard monotherapy dose oftemozolomide.

The effective amount of 2,4-ds-PBN may be from about 0.005 w/w % toabout 0.1 w/w % of the diet being administered. The glioma may be anastrocytoma, an oligodendroglioma, or a glioblastoma multiforme, or aTGF-β1-, MGMT- and/or APGN-expressing form of any of the foregoing. Themethod may further comprise assessing expression of LBP, wherein reducedLBP levels as compared to untreated controls indicates improvedprognosis.

In still another embodiment, there is provided a method for inhibitingglioma recurrence comprising (a) identifying a subject having a gliomaand (b) administering to said subject doses of (i) 2,4-disulfonyl phenyltert-butyl nitrone (2,4-ds-PBN), and (ii) temozolomide effective toinhibit the recurrence of said glioma. Administration may be through aroute requiring subsequent passage of 2,4-ds-PBN across the blood brainbarrier, such as enteral, intravenous, or intra-arterial.

The subject may have a recurrent or metastatic glioma, and or may havepreviously failed one or more anti-glioma therapies. The effective doseof 2,4-ds-PBN may be from about 5 to about 150 mg/kg body weight perday. The enteral administration may be through dietary supplementationof a food component, or the enteral administration may be in the form ofa pill or a liquid. The subject may be a human.

The method may further comprise a secondary anti-glioma therapy, such asradiation, surgery, or chemotherapy, such as temozolomide, lomustine,vincristine, matulane, PCV, BCNU, CCNU and/or DFMO. The secondaryanti-glioma agent may be temozolomide, and the effective dose oftemozolomide may be lower than the standard monotherapy dose oftemozolomide.

The effective amount of 2,4-ds-PBN may be from about 0.005 w/w % toabout 0.1 w/w % of the diet being administered. The glioma may be anastrocytoma, an oligodendroglioma, or a glioblastoma multiforme, or aTGF-β1-, MGMT- and/or APGN-expressing form of any of the foregoing. Themethod may further comprise screening for glioma formation in saidsubject. The method may further comprise assessing efficacy by measuringthe expression of liposaccharide binding protein prior to and after atreatment with 2,4-ds-PBN. The method may further comprise assessingexpression of LBP, wherein reduced LBP levels as compared to untreatedcontrols indicates improved prognosis.

In still yet another embodiment, there is provided a method of detectingglioblastoma comprising (a) obtaining a sample from a subject; and (b)assessing liposaccharide binding protein (LBP) level in said sample,wherein an LBP level higher than that from a comparable normal controlsample indicates the presence of glioblastoma in said subject. AssessingLBP level may comprise immunologic assessment or nucleic acidassessment, such as immunologic assessment selected from ELISA, RIA,Western blot, or immunohistochemistry, or nucleic acid assessmentselected from RT-PCR, Northern blot, RNA-Seq or microarray. The samplemay be whole blood, serum, plasma or urine.

A further embodiment comprises a method of monitoring glioblastomaprogression comprising (a) obtaining a sample from a subject; (b)assessing liposaccharide binding protein (LBP) level in said sample; and(c) repeating steps (a) and (b) at a second time point, wherein an LBPlevel higher in step (c) as compared to step (b) indicates progressionof glioblastoma in said subject. Assessing LBP level may compriseimmunologic assessment or nucleic acid assessment, such as immunologicassessment selected from ELISA, RIA, Western blot, orimmunohistochemistry, or nucleic acid assessment selected from RT-PCR,Northern blot, RNA-Seq or microarray. The sample may be whole blood,serum, plasma or urine.

Yet a further embodiment comprises a method of staging glioblastomacomprising (a) obtaining a sample from a subject; (b) assessingliposaccharide binding protein (LBP) level in said sample; (c) comparingthe LBP level of step (b) with a control samples for low, mid and/orhigh grade glioblastomas, and (d) assigning a grade to said glioblastomain said subject. Assessing LBP level may comprise immunologic assessmentor nucleic acid assessment, such as immunologic assessment selected fromELISA, RIA, Western blot, or immunohistochemistry, or nucleic acidassessment selected from RT-PCR, Northern blot, RNA-Seq or microarray.The sample may be whole blood, serum, plasma or urine.

An additional embodiment comprises a method of treating a subject withglioblastoma comprising administering to said subject a therapeuticagent linked to a liposaccharide binding protein (LBP) targeting agent.The LBP targeting agent may be an antibody, ScFv, Fab or F(ab′)₂ orpeptide. The therapeutic agent may be a chemotherapeutic agent, aradiotherapeutic agent, immunotherapeutic agent or biological agent. Thechemotherapeutic agent may be temozolomide or 2,4-disulfonyl phenyltert-butyl nitrone (2,4-ds-PBN). The glioblastoma may be drug resistant,such as temozolomide resistant. The glioblastoma may have been treatedpreviously with temozolomide and/or 2,4-disulfonyl phenyl tert-butylnitrone (2,4-ds-PBN). The glioblastoma may be recurrent and/ormetastatic. The linkage of the therapeutic agent to LBP may becleavable. The method may further comprise assessing LBP expression insaid patient or said glioblastoma.

Yet an additional embodiment comprises a method of identifying aglioblastoma boundary in a subject comprising (a) administering to saidsubject an imaging agent linked to liposaccharide binding protein; and(b) imaging a glioblastoma site in said subject. The method may furthercomprise administering to said subject an imaging agent linked to ELTD1,Slit-3 or Spondin-1. The imaging agent may be a dye, a radiolabel, afluorescent label, a chemiluminescent label, an MRI label or a nearinfrared label. The method may further comprise excising saidglioblastoma following imaging, optionally further comprising re-imagingsaid glioblastoma site after excising.

An even further embodiment comprises a method of identifying aglioblastoma cell in a tissue sample from a subject comprising (a)obtaining a tissue sample from said subject (b) contacting said tissuesample with a label linked to liposaccharide binding protein; and (c)detecting said label-LBP conjugate bound to said tissue sample. Themethod may further comprising contacting said tissue sample with a labellinked to said subject an imagine agent linked to ELTD1, Slit-3 orSpondin-1. The label may be a dye, a radiolabel, a fluorescent label, achemiluminescent label, an MRI label or a near infrared label. Thetissue sample may be an unfixed fresh biopsy sample. The tissue samplemay be a fixed biopsy sample.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions and kits of theinvention can be used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “contain” (and any form of contain, such as “contains” and“containing”), and “include” (and any form of include, such as“includes” and “including”) are open-ended linking verbs. As a result, adevice or a method that “comprises,” “has,” “contains,” or “includes”one or more elements possesses those one or more elements, but is notlimited to possessing only those one or more elements or steps.Likewise, an element of a device or method that “comprises,” “has,”“contains,” or “includes” one or more features possesses those one ormore features, but is not limited to possessing only those one or morefeatures.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 : Percent animal survival Kaplan-Meier curve for untreated, andTMZ-, OKN-007 and combined OKN-007 and TMZ-treated G55 glioma-bearingmice.

FIG. 2 : In vivo tumor volumes (mm³) obtained at days 19-22 followingMRI detection of tumors (>5 mm³).

FIG. 3 : MR images depicting tumors in the mid-tumor region (maximaltumor) for each treatment group (TMZ, OKN-007 (01(N), combined OKN-007and TMZ (Comb), or untreated (UT).

FIG. 4 : Tumor volumes obtained at the last MRI time-point for eachtreatment group (UT, TMZ, OKN or combined treatments).

FIG. 5A: Tumor volumes (means±S.D.) obtained at multiple time-points foreach treatment group (UT: black open circle with dash-dot line; TMZ:blue open square with a dash line; OKN: green open upward triangle witha dot line; Combined: red closed downward triangle with a solid line).The treatment window initiated when tumors were >10 mm³ for up to >50days.

FIG. 5B: Changes (rCBF in tumors at 21-22 days following tumor detectionminus rCBF at initial tumor detection) in normalized (normalized tocontralateral or normal brain tissue) rCBF values in UT, TMZ, OKN orcombined treated G55 glioma-bearing mice.

FIG. 6 : Ex vivo LBP levels measured from ELISA in tumor tissue lysatesobtained from UT, TMZ, OKN or combined treated G55 glioma-bearing mice.

FIG. 7 : Ex vivo LBP levels measured from ELISA in blood sera obtainedfrom UT, TMZ, OKN or combined treated G55 glioma-bearing mice.

FIG. 8 : In vitro IC50 assessment of TMZ-resistant (T98G, G55) andTMZ-sensitive (U251) GBM cell lines combined with OKN-007.

FIG. 9 : OKN-007 effect on LBP in glioma-bearing rat tumors. ELISAassessment of LBP performed on tissue lysates from F98 glioma-bearinganimals showed expression differences during the therapy with OKN-007.Levels of LBP in the tumor bearing animals were significantly elevatedcompared to the non-tumor control group, and OKN-007 treatment broughtthe levels of LBP close to the non-tumor controls.

FIG. 10 : OKN-007 effect in LBP in glioma-bearing rat sera. ELISAassessment of LPB using the blood serum from the same animals from FIG.9 confirmed that LPB can serve as a serum biomarker that can predict thetherapy outcome of OKN-007 in gliomas.

FIGS. 11A-C: LBP levels by tumor grade. LBP level is elevated inhigh-grade human patient gliomas compared with tumors classified aslow-grade gliomas. Representative immunohistochemistry staining for LBPin high-grade glioma: glioblastoma multiforme (FIG. 11A), and inlow-grade astrocytoma (FIG. 11B). (FIG. 11C) Immunohistochemistry (IHC)score means of LBP expression in high-grade (GBM-glioblastomas,AA-anaplastic astrocytomas, AO-anaplastic oligodendrogliomas; 94 patienttissue samples) and low-grade (LGA-low grade astrocytomas, oligo-benignoligodendrogliomas; 45 patient tissue samples) human gliomas. Gradingcriteria: 0: 0%; 1: 0 to <25%; 2: 25 to <50%; 3: 50 to <75%; 4: 75-100%detection of IHC stain.

FIG. 12 : LBP as a marker for OKN efficacy. For each treatment group,LBP levels were assessed by ELISA from blood and tumor tissue samples atthe end of the study. All treatments were found to significantlydecrease LBP in blood compared to UT mice with G55 gliomas. OKN andcombined treatments were found to significantly decrease LBP in tumortissue compared to UT mice with G55 gliomas. In tumor tissues or blood,there seemed to be no difference between OKN or combined therapies onLBP levels.

FIGS. 13A-B: (FIG. 13A) Percent animal survival Kaplan-Meier curve foruntreated (UT), and TMZ-, OKN-007 and combined OKN-007 and TMZ-treatedG55 glioma-bearing mice. All treatment groups were found to have asignificantly higher survival (p<0.05 or more), compared to UTG55-glioma-bearing mice. The combined treatment group was found to havea significantly higher percent survival (p<0.01) than the TMZ-treatedgroup. (FIG. 13B) In vivo tumor volumes (mm³) obtained at days 19-22following MRI detection of tumors (>5 mm³). All treatment groups werefound to have significantly lower tumor volumes (p<0.01 or more),compared to the UT group.

FIGS. 14Ai-Diii: Representative MR images depicting tumors in themid-tumor region (maximal tumor) for (FIG. 14A) untreated (UT), or eachtreatment group (FIG. 14B) TMZ, (FIG. 14C) OKN-007 (OKN), or (FIG. 14D)combined OKN-007 and TMZ (Comb), at 19-22 days following tumordetection. For images labeled “i”, the tumors are highlighted with afaint line to depict tumor boundaries. Images in panels “ii” or “iii”are other examples in treatment groups A-D, depicting either consistencyin the UT group, or variability in the treatment groups. There were nodetectable tumors in FIG. 14Ciii or FIG. 14Diii for the OKN-007- orcombined-therapy groups, following treatment at this time-point range.

FIGS. 15Ai-E: Vascular changes as depicted by perfusion MRI [rCBF(relative cerebral blood flow) in tumors at 21-22 days following tumordetection minus rCBF at initial tumor detection] in normalized(normalized to contralateral or normal brain tissue) rCBF values for(FIG. 15A) UT, (FIG. 15B) TMZ, (FIG. 15C) OKN or (FIG. 15D) combinedtreated G55 glioma-bearing mice. The top panel of images labeled “i” arerepresentative T2-weighted morphological MR images for each treatmentgroup, whereas the images labeled “ii” are representative perfusion mapsfor each treatment group. (FIG. 15E) Quantitative assessment of a changein normalized rCBF in UT and TMZ-, OKN-, or combined therapy-treated G55glioma-bearing mice. All treatment groups had a significantly lower(p<0.05 or more) change in normalized rCBF, compared to the UT group.

FIG. 16 : Gene-fold changes when comparing TMZ+OKN to TMZ alonetreatment groups in LN18 GBM cells.

FIG. 17 : Gene-fold changes when comparing TMZ+OKN to TMZ alonetreatment groups in LN229 GBM cells.

FIG. 18 : Migration speeds of G55 GBM cells in PDMS(polydimethylsiloxane) micro-channels coated with laminin, eitheruntreated (UT), or treated with either OKN-007 (OKN), TMZ or bothOKN+TMZ, at 22, 28 and 46 h post-treatment. There was a significantdifference between UT cells (*) and those treated with OKN or OKN+TMZ at22 and 28 h, and with all treatment groups at 46h. There was asignificant difference between TMZ and OKN+TMZ groups (†) at 22 and 28h. *(†) p<0.05, **(††) p<0.01, *** p <0.001.

FIG. 19 : Microarray analysis of mRNA samples from OKN-007-treated anduntreated rat F98 glioma-bearing tumors, indicating down-regulated genes(green; >2-fold change) in the treated group (n=4) compared to theuntreated group (n=4) in a network pathway schematic. Majordown-regulated gene pathways affected include TGFβ1, PDGFBB, P38 MAPK,NFKB, some MMPs (particularly MMP12), DCN (decorin), SERPINB2, LUM, LBP(lipopolysaccharide binding protein), and several collagens.

FIG. 20 : TGFβ1 as a master regulator of 57 down-regulated genes byOKN-007 in rat F98 treated gliomas vs. untreated tumors. (UpstreamRegulator Analysis, IPA). OKN-007 down-regulates 57 genes includingcollagens, MMP12 (tissue remodeling), SERPINB2 (serpin peptidaseinhibitor), IGFBP5 (insulin-like growth factor binding protein).

FIGS. 21A-C: TGFβ1 IHC of rat F98 orthotopic tumors (mid-tumor region)either (FIG. 21A) untreated, or (FIG. 21B) treated with OKN-007. (FIG.21C) ELISA TGFβ1 protein levels (pg/mL) in untreated (UT) or OKN-007(OKN)-treated from F98 tumor tissue lysates.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The prognosis of patients who are diagnosed with glioblastoma multiformeis very poor, due to the difficulty of an early and accurate diagnosisand the lack of currently efficient therapeutic compounds. In addition,gliomas that are initially sensitive to drugs can become resistant overtime, thereby presenting an additional challenge to treating this highlylethal form of cancer.

As mentioned above, 2,4-ds-PBN, a structural analog of PBN, was able todecrease tumor volumes and the delay in tumor growth rate of animalswith gliomas, and also significantly increased survival rate. However,it is unknown whether 2,4-ds-PBN could positively affect the outcome oftemozolomide (TMZ)-based therapies, including prevention and overcomingof TMZ-resistance in glioma cells. Given the standard-of-care status ofTMZ treatment, and the prevalence of TMZ resistance, such a benefit thiswould be of considerable significance to patients undergoing treatmentwith TMZ.

Thus, the inventor sought to look at the effects of 2,4-ds-PBN in thecontext of TMZ resistance and co-therapy. In vitro cell data clearlyindicates that 2,4-ds-PBN significantly decreases the cell viability ofTMZ-resistant GBM cells treated with TMZ. The in vivo data clearly showsthat combined 2,4-ds-PBN and TMZ treatment substantially increasesanimal survival and decreases tumor volumes in a TMZ-resistant gliomaxenograft nude mouse model. These unexpected results point towards thecombined use of 2,4-ds-PBN and TMZ as a powerful new therapeutic regimenfor gliomas.

These and other aspects of the disclosure are described in detail below.

2. ANAPLASTIC GLIOMA STRATA

A. Clinical Features

The anaplastic gliomas are intermediate grade infiltrativegliomas—classified between low (localized, slow growing) andglioblastoma multiforme (rapidly growing and highly invasive).Anaplastic astrocytomas (AA) are tumors that arise from brain cellscalled astrocytes and/or their precursors. Astrocytes are support cellsof the central nervous system. The majority of astrocytic tumors inchildren are low-grade, whereas the majority in adults are high-grade.These tumors can occur anywhere in the brain and spinal cord.

Oligodendrogliomas are gliomas derived from oligodendrocytes and/ortheir precursors. Oligodendrocytes that have a role in the structure andfunction of myelinated neurons in the brain. Anaplasticoligodendroglioma (AO) are more aggressive than oligodendrogliomas, butare also more sensitive to chemotherapy than are anaplasticastrocytomas. A high rate of response to the use of PCV (procarbazine,CCNU, vincristine) chemotherapy has led to the common use of PCVchemotherapy prior to radiation therapy, following irradiation, and/orat tumor recurrence and progression. Another glioma appears ashistologic mixture of both oligodendroglioma and astrocytoma tumor formsand is called oligoastrocytoma. While oligoastrocytoma can be low-grade,the majority of the mixed oligoastrocytomas are anaplasticoligoastrocytomas (AOA).

The last glioma subgroup are ependymomas. One subtype of malignantependymomas is the anaplastic ependymoma (AE); these tumors arise fromependymal cells and/or their precursors that line the cerebrospinalfluid passageways, called ventricles. These tumors are classified aseither supratentorial (in the top part of the head) or infratentorial(in the back of the head).

Clinical features and symptoms produced by gliomas depend on thelocation of the tumor and the age of the patient. The most commonlocation for gliomas is in the cerebral hemispheres in adults and thecerebellum, brainstem, hypothalamus, and thalamus in children.

Spinal cord gliomas are much less common then gliomas of the brain.Patients with these tumors have symptoms that vary depending on locationin the brain or spinal cord. They can produce symptoms of headache,seizures, nausea and vomiting, limb weakness, unilateral sensorychanges, personality change, and unsteadiness in walking.

B. Classifications

Anaplastic Astrocytoma. The histologic features of anaplasticastrocytomas are similar to those of low-grade astrocytomas but thesefeatures are more abundant and exaggerated. These tumors are WHO gradeIII (Kleihues et al., 1993; Kleihues and Cavenee, 2000). Cellularity ismore increased, as are nuclear and cellular pleomorphism. These featuresmay be extreme, with back-to-back cells and bizarre, hyperchromaticnuclei. Cytoplasm may be scanty, with nuclear lobation and enlargementindicating anaplasia. Mitotic activity is easily recognized in mostanaplastic astrocytomas but inexplicably may be absent in areas withgemistocytes.

The range of anaplasia in this grade is broad, with some examplesshowing low cellularity and pleomorphism with a few mitotic figures andothers being highly cellular and pleomorphic with frequent mitoses,lacking only the necrosis required for a histologic diagnosis ofglioblastoma. For this reason, it is useful to have a more objectiveindicator of behavior, and some markers of cell proliferation have beenused in an attempt to predict prognosis more accurately. The most usedmarkers in this area have been antibodies to bromodeoxyuridine (BrdU)and Ki-67 (Davis et al., 1995). The cellular incorporation of BrdU is aspecific marker of the DNA synthesis phase of the cell cycle, whereasthe Ki-67 antibody labels an antigen that is present in all phases ofthe cell cycle except Go. Both antibodies can be identified byimmunohistochemical staining in paraffin-embedded tissue sections. As ageneralization, higher labeling rates for anaplastic astrocytomas isassociated with poor prognosis (Hoshino et al., 1993; Davis et al.,1995; Lamborn et al., 1999).

Glioblastoma multiforme. Glioblastoma, also known as glioblastomamultiforme, is the glioma with the highest grade of malignancy, WHOgrade IV (Kleihues and Cavenee, 2000). It represents 15% to 23% ofintracranial tumors and about 50%-60% of astrocytomas. Most examples aregenerally considered to arise from astrocytes because glial fibrillaryacidic protein can be identified in the cell cytoplasm. Some examples,however, apparently arise from other glial lineages, such asoligodendrocytes. Glioblastoma is the most frequently occurringastrocytoma. Autopsy and serial biopsy studies have shown that someastrocytomas progress through the grades of malignancy withtransformation from low-grade to anaplastic astrocytoma to glioblastoma(Muller et al., 1977). But, because some examples of glioblastoma appearto arise rapidly in otherwise normal patients and are recognized whenthey are small, it is thought that this variety of glioblastoma can alsoarise directly from malignant transformation of astrocyte precursorcells without passing through the lower grades of malignancy (Kleihuesand Ohgaki, 1997; 1999).

Tumor necrosis is the characteristic gross feature that distinguishesglioblastoma from anaplastic astrocytoma (Nelson et al., 1983; Burger etal., 1985; 1991). Another microscopic feature that is distinctive anddiagnostic is the presence of proliferative vascular changes within thetumor. These changes may occur in the endothelial cells (vascularendothelial hyperplasia or proliferation) or in the cells of the vesselwall itself (vascular mural cell proliferation). Both types of changeare sometimes considered together as microvascular proliferation.Glioblastomas cellularity is usually extremely high. The individualcells may be small, with a high nuclear:cytoplasmic ratio, or very largeand bizarre, with abundant eosinophilic cytoplasm. These same smallcells may appear to condense in rows around areas of tumor necrosis,forming the characteristic pseudopalisades. Glioblastoma tumors have apropensity to infiltrate the brain extensively, spreading even todistant locations and giving the appearance of a multifocal glioma. Someexamples are truly multifocal (i.e., arising in multiple simultaneousprimary sites) while many of these multifocal tumors show a histologicconnection when the whole brain is examined at autopsy.

Oligodendrogliomas. Like astrocytomas, oligodendrogliomas mimic thehistology of their presumed cell of origin. They also arise primarily inthe white matter but tend to infiltrate the cerebral cortex more than doastrocytomas of a similar grade of malignancy. Like astrocytomas,grading schemes of histologic malignancy have been used foroligodendrogliomas, but these correlate less well with prognosis thanthose used for astrocytomas (Burger et al., 1987; Bigner et al., 1998;Daumas-Duport et al., 1997). Many of the histologic features used tograde oligodendrogliomas are similar to those used for astrocytomas:cellularity, pleomorphism, mitotic activity, vascular changes, andnecrosis. Lower-grade oligodendrogliomas may have microcysts.Oligodendrogliomas of all histologic grades tend to infiltrate thecortex readily and to form clusters of neoplastic cells in the subpialregion, around neurons, and around blood vessels. In general, the cellsof oligodendrogliomas have round, regular nuclei and distinctcytoplasmic borders with clearing of the cytoplasm. Another fairlydistinctive and diagnostically helpful feature is the vascular patternof oligodendrogliomas, referred to as “chicken-wire” vessels that candivide the tumor into discrete lobules. With increasing anaplasia,oligodendrogliomas can become highly cellular and pleomorphic,approaching an appearance of glioblastoma multiforme with the presenceof necrosis. Although it is correct to classify these as anaplasticoligodendrogliomas, some would use the term glioblastoma once necrosisis identified in any high-grade glial neoplasm. One justification forseparating anaplastic oliogdendrogliomas from astrocytic glioblastomasis the slightly better prognosis of the former, even in this highestgrade of malignancy. Some authors have reported that a MIB-1 labelingindex of >3%-5% predicts a worse prognosis in oligodendrogliomas(Heegard et al., 1995; Kros et al., 1996; Dehghani et al., 1998).

Oligoastrocytomas. Many, if not most, oligodendrogliomas occur with aregional or intimate cellular mixture of astrocytoma. For the diagnosisof mixed glioma, the proportion of each should be substantial, butauthors have differing opinions with respect to exact numbers;

usually a mixture with a range from 10% to 25% of the minor element isused to diagnose a mixed glioma. Oligoastrocytomas and anaplasticoligoastrocytomas correspond to WHO grade II or grade III, respectively(Kleihues and Cavenee, 2000). Histologic features of anaplasia may bepresent in either component and will affect the prognosis adversely.Such features include marked cellular pleomorphism, high cellularity,and a high mitotic rate. Microvascular proliferation and necrosis mayalso be seen. Prognosis and response to therapy have not been shown todepend on the proportion of the oligodendroglial versus the astrocyticcomponent (Shaw et al., 1994), although paradoxically, the BrdU LI ofthe oligodendroglial component is more predictive for survival than theastrocytic component (Wacker et al., 1994) and far advanced tumorprogressions are dominated by the astrocytic component.

3. PHENYL N-TERT-BUTYL NITRONES (PBNS) AND TEMOZOLOMIDE (TMZ)

A. PBN's

The compound phenyl N-tert-butyl nitrone (PBN) was first synthesized inthe 1950's, but in 1968 it was discovered to be very useful to trap andstabilize free radicals in chemical reactions and hence it was termed aspin-trap (Janzen, 1971). Although PBN is the prototype spin-trap,several other nitrones have been synthesized and found useful to trapand characterize free radicals in chemical reactions. These spin trapswere used in chemical reactions first, but in the mid-1970's they beganto be used to trap free radicals in biochemical and biological systems(Poyer et al., 1978). Pharmacokinetic studies have shown that PBN isreadily and rapidly distributed almost equally to all tissues, has ahalf-life in rats of about 132 minutes and is eliminated mostly in theurine. Relatively few metabolism studies have been done, but it is knownthat some ring hydroxylation (primarily in the para position) of thecompound occurs in the liver.

Novelli first showed that PBN could be used to protect experimentalanimals from septic shock (Novelli et al., 1986), and indeed this waslater confirmed by other groups (Pogrebniak et al., 1992). The use ofPBN and derivations as pharmacological agents began after discoveries in1988 that showed that PBN had neuroprotective activity in experimentalbrain stroke models (Floyd, 1990; Floyd et al., 1996; Carney et al.,1991). These results were repeated and extended, (see Clough-Helfman etal., 1991; Cao et al., 1994; Folbergrova et al., 1995; Pahlmark et al.,1996). Others have summarized the extensive neuroprotectivepharmacological research effort on PBN and derivatives (Floyd, 1997;Hensley et al., 1996). In addition to neurodegenerative diseases, PBNhas been shown to protect in other pathological conditions whereROS-mediated processes are involved, including diabetes and many otherconditions. The mechanistic basis of why PBN and some of its derivativesare so neuroprotective in experimental stroke and several otherneurodegenerative models has not been completely elucidated yet.However, it is clear that its action cannot simply be explained by itsability to trap free radicals.

The general formula for PBNs is:

wherein:

X is phenyl or

R is H,

and n is a whole integer from 1 to 5; or

Y is a tert-butyl group that can be hydroxylated or acetylated on one ormore positions; phenyl; or

wherein W is

or Z; and Z is a C₁ to C₅ straight or branched alkyl group.

B. PBN's in Cancer

U.S. Pat. No. 5,569,902 (incorporated herein by reference) describes theuse of nitrone free radical trapping agents for the treatment of cancer.Specifically, PBN and related compounds are described as being useful inthe preparation of an anti-carcinogenic diet and the preparation of suchsupplemented diets. Those subjects most likely to beneficially receivethe nitrones would include: (1) those having had pretumor testsindicating a high probability of the presence of tumors, (2) thoseexposed to very potent carcinogenic environments and their probabilityof tumor progression is high, and (3) to those whose geneticpredisposition makes their likelihood of tumor development high.

U.S. Patent Publication 2007/0032453 (incorporated herein by reference)describes the effect of the anti-inflammatory phenyl N-tert-butylnitrones (PBNs) on gliomas using MRI techniques. PBN itself was able tocontrol tumor development when provided to a subject either before, atthe time of or after tumor implantation. Thus, it was proposed to usePBN, and related nitrone free radical trapping agents, as therapeuticagents for gliomas.

C. 2,4-Disulfonyl Phenyl N-Tert-Butyl Nitrone (2,4-ds-PBN)

U.S. Pat. No. 5,488,145 (incorporated herein by reference) describes2,4-disulfonyl phenyl-tert-butyl nitrone and its pharmaceuticallyacceptable salts. These materials were described as usefulpharmaceutical agents for oral or intravenous administration to patientssuffering from acute central nervous system oxidation as occurs in astroke or from gradual central nervous system oxidation which canexhibit itself as progressive central nervous system function loss.

-   -   2,4-disulfonyl PBN (also referred to as OKN-0007)

2,4-disulfonyl PBN's two sulfonate groups was expected to exhibitimproved water solubility, but was also expected to exhibit poortransport across the blood/brain barrier because of its lipophobiccharacter. However, when the present compound was made and tested invivo, it showed an unexpected increase in efficacy as compared to PBN.This increase in efficacy occurred along with an increase in potency ascompared to PBN. In direct contrast to this marked increase in potencyand efficacy there was a marked and highly significant decrease intoxicity as compared to PBN.

These results were unexpected because in the general literature onstructure/activity relationships within specific defined families ofcompounds therapeutic potency typically covaries with toxicity. Thus,most related compounds maintain their ratio of therapeutic potency totoxicity. In contrast, the compound of this invention deviates from thisexpected relationship when its potency increased and its toxicitydecreased relative to closely related analogs.

Accordingly, in one aspect, the invention provides the PBN-disulfonylcompound and its pharmaceutically acceptable salts. In a second aspect,the invention provides intravenously- and orally-administrablepharmaceutical compositions having this compound or its salt as activeingredient.

-   -   2,4-ds PBN may exists at higher pHs in an ionized salt form:

orwhere X is a pharmaceutically acceptable cation. Most commonly, thiscation is a monovalent material such as sodium, potassium or ammonium,but it can also be a multivalent alone or cation in combination with apharmaceutically acceptable monovalent anion, for example calcium with achloride, bromide, iodide, hydroxyl, nitrate, sulfonate, acetate,tartrate, oxalate, succinate, palmoate or the like anion; magnesium withsuch anions; zinc with such anions or the like. When these combinationsof a polyvalent cation and a monovalent anion are illustrated instructural formulae, herein, the monovalent anion is identified as “Y.”

Among these materials, the free acid and the simple sodium, potassium orammonium salts are most preferred with the calcium and magnesium saltsalso being preferred but somewhat less so.

2,4-ds PBN can be prepared by a two-step reaction sequence. In the firststep, commercially available tertiary butyl nitrate(2-methyl-2-nitropropane) is converted to the corresponding n-hydroxylamine using a suitable catalyst such as an activated zinc/acetic acidcatalyst or an aluminum/mercury amalgam catalyst. This reaction can becarried out in 0.5 to 12 hours and especially about 2 to 6 hours or soat a temperature of about 15-100° C. in a liquid reaction medium such asalcohol/water mixture in the case of the zinc catalyst or an ether/watermixture in the case of the aluminum amalgam catalyst.

In the second step, the freshly formed hydroxylamine is reacted with4-formyl-1,3-benzenedisulfonic acid, typically with a slight excess ofthe amine being used. This reaction can be carried out at similartemperature conditions. This reaction is generally complete in 10 to 24hours. The product so formed is the free acid and is characterized by amolecular weight of 89 g/mole. It is a white powdery material whichdecomposes upon heating. It is characterized by a solubility in water ofgreater than 1 gram/ml and a ¹H NMR spectrum in D₂ O of 8.048 ppm (dd,8.4, 1.7 Hz); 8.836 ppm (d, 8.4 Hz); 8.839 ppm (d, 1.7 Hz); 8.774 ppm(s).

The various salts can be easily formed by admixing the free acid inaqueous medium with two equivalents of the appropriate base, forexample, KOH for the potassium salt, and the like.

One synthesis is based on the work by R. H. Hinton and E. G. Janzen (J.Org. Chem. 57:2646-2651, 1992). It involves the condensation of analdehyde with a hydroxylamine. The hydroxylamine is unstable and isprepared fresh on the day of use using an activated zinc catalyst. Thesynthesis is as follows.

TABLE 1 Prerequisite Chemicals 1. 95% Ethanol 2. 2-Methyl-2-nitropropane3. Zinc dust 4. Glacial acetic acid 5. Diethyl ether 6. Saturated sodiumchloride 7. Magnesium Sulfate, Anhydrous solid 8.4-Formyl-l,3-benzenesulfonic acid (MW 310.21 g/mole), disodium salt,hydrate 9. Methanol 10. Dichloromethane

TABLE 2 Preparation of N-t-Butylhydroxylamine 1. A 500 mL three neckround bottom flask is equipped with a magnetic stir bar, thermometeradapter, thermometer, and addition funnel. 2. 95% ethanol (350 mL) wasadded to the flask and cooled to 10° C. in an ice bath. 3.2-Methyl-2-nitropropane (6.18 g, 0,060 mole), and zinc dust (5.89 g,0,090 mole) were added in single portions. 4. Glacial acetic acid (10.8g, 0, 180 mole) was placed in the addition funnel and added dropwise atsuch a rate with vigorous stirring to maintain the temperature below 15°C. 5. The ice bath was removed and mixture was stirred for 3 hrs at roomtemperature. 6. The solvent was stripped from the mixture, leavingt-butylhydroxylamine, zinc acetate, and water. 7. Dichloromethane (50mL) was added and the mixture filtered through a Buchner funnel. 8. Thezinc acetate cake left on the filter paper was washed with 2X 25 mLdichloromethane. 9. Water was separated from the filtrate in aseparatory funnel and the organic layer dried over magnesium sulfate.10. The magnesium sulfate was removed by filtering through fluted filterpaper, then dichloromethane stripped off by rotary evaporation. 11. Theproduct (100% yield = 5.34 g), a viscous liquid, was dissolved inmethanol (50 mL) for use below.

TABLE 3 Preparation of 2,4-disulfonylphenyl-N-t-butylnitrone 1. A 3-neck250 ml round bottom flask was set up with a stir bar, a gas dispersiontube, an addition funnel, and a Friedrichs condenser cooled withrecirculating ice water. 2. To the flask were added 200 mL of methanol,4-formyl-1,3 -benzenedisulfonic acid (9.31 g, 30 mmoles) andN-t-butylhydroxylamine (25 mL of the methanol solution from part A, 30mmoles theoretical). 3. The reaction was heated to reflux with a heatingmantle while bubbling the reaction with nitrogen with stirring. 4. Themixture was refluxed for 2 hours. 5. The remainder of hydroxylamine fromabove was added. 6. Refluxing was continued with nitrogen bubbling forat least 18 hours, but not more than 24 hours. 7. The hot reactionmixture was filtered on a Buchner funnel, and the solid washed with hotmethanol. 8. The methanol was stripped off by rotary evaporation to ayellow, viscous oil. 9. Hot 1:1 ethanol:acetone (200 mL) was added andthe mixture heated to dissolve the oil. 10. The solution was cooled tocrystallize the product. 11. The product was collected on a Buchnerfunnel and dried under vacuum overnight. 12. The reaction typicallygives 75% yield of a white powder.Other methods of synthesis are disclosed in the prior art as well.

D. Temozolomide

Temozolomide (TMZ; brand names Temodar® and Temodal® and Temcad®) is anoral chemotherapy drug. It is an alkylating agent used as a treatment ofsome brain cancers; as a second-line treatment for astrocytoma and afirst-line treatment for glioblastoma multiforme. TMZ is a prodrug andan imidazotetrazine derivative of the alkylating agent dacarbazine. Itsapproved indications include nitrosourea- and procarbazine-refractoryanaplastic astrocytoma, and newly diagnosed glioblastoma multiforme. TMZhas been available in the U.S. since August 1999, and in other countriessince the early 2000s.

The most common side effect from TMZ is bone marrow suppression. Themost common non-hematological adverse effects associated withtemozolomide are nausea and vomiting, which are either self-limiting orreadily controlled with standard antiemetic therapy. These lattereffects are usually mild to moderate (grade 1 to 2). The incidence ofsevere nausea and vomiting is around 4% each. Patients who havepre-existing or a history of severe vomiting may require antiemetictherapy before initiating temozolomide treatment. Temozolomide should beadministered in the fasting state, at least one hour before a meal.Antiemetic therapy may be administered before, or following,administration of temozolomide. Temozolomide is contraindicated inpatients with hypersensitivity to its components or to dacarbazine. Theuse of temozolomide is not recommended in patients with severemyelosuppression. A standard oral dose of temozolomide would be 150mg/m² daily, increased to 200 mg/m² daily for maintenance. Lower levelswould be anything below standard dosing, including 125 mg/m², 100 mg/m²,75 mg/m², or 50 mg/m², for example.

Temozolomide is genotoxic, teratogenic and fetotoxic and should not beused during pregnancy. Lactating women should discontinue nursing whilereceiving the drug because of the risk of secretion into breast milk.One study indicated that women that have taken temozolomide withoutconcomitant fertility preservation measures achieve pregnancy to alesser rate later in life, but the study was too small to showstatistical significance in the hypothesis that temozolomide wouldconfer a risk of female infertility. In male patients, temozolomide canhave genotoxic effects. Men are advised not to father a child during orup to six months after treatment and to seek advice on cryoconservationof sperm prior to treatment, because of the possibility of irreversibleinfertility due to temozolomide therapy. Very rarely temozolomide cancause acute respiratory failure or liver damage. The therapeutic benefitof temozolomide depends on its ability to alkylate/methylate

DNA, which most often occurs at the N-7 or O-6 positions of guanineresidues. This methylation damages the DNA and triggers the death oftumor cells. However, some tumor cells are able to repair this type ofDNA damage, and therefore diminish the therapeutic efficacy oftemozolomide, by expressing a protein O⁶-alkylguanine DNAalkyltransferase (AGT) encoded in humans by the O-6-methylguanine-DNAmethyltransferase (MGMT) gene. In some tumors, epigenetic silencing ofthe MGMT gene prevents the synthesis of this enzyme, and as aconsequence such tumors are more sensitive to killing by temozolomide.Conversely, the presence of AGT protein in brain tumors predicts poorresponse to temozolomide and these patients receive little benefit fromchemotherapy with temozolomide.

Laboratory studies and clinical trials have investigated the possibilityof increasing the anticancer potency of temozolomide by combining itwith other pharmacologic agents. For example, clinical trials haveindicated that the addition of chloroquine might be beneficial for thetreatment of glioma patients. Laboratory studies found that temozolomidekilled brain tumor cells more efficiently when epigallocatechin gallate(EGCG), a component of green tea, was added; however, the efficacy ofthis effect has not yet been confirmed in brain-tumor patients.Preclinical studies reported in 2010 on investigations into the use ofthe novel oxygen diffusion-enhancing compound trans sodium crocetinate(TSC) when combined with temozolomide and radiation therapy and aclinical trial was underway as of August 2015.

While the above-mentioned approaches have investigated whether thecombination of temozolomide with other agents might improve therapeuticoutcome, efforts have also started to study whether altering thetemozolomide molecule itself can increase its activity. One suchapproach permanently fused perillyl alcohol, a natural compound withdemonstrated therapeutic activity in brain cancer patients, to thetemozolomide molecule. The resultant novel compound, called NEO212 orTMZ-POH, revealed anticancer activity that was significantly greaterthan that of either of its two parent molecules, temozolomide andperillyl alcohol. Although, as of 2016, NEO212 has not been tested inhumans, it has shown superior cancer therapeutic activity in animalmodels of glioma, melanoma, and brain metastasis of triple-negativebreast cancer.

Because tumor cells that express the MGMT gene are more resistant to theeffects of temozolomide, researchers investigated whether the inclusionof O⁶-benzylguanine (O⁶-BG), an AGT inhibitor, could overcome thisresistance and improve the drug's therapeutic effectiveness. In thelaboratory, this combination indeed showed increased temozolomideactivity in tumor-cell culture in vitro and in animal models in vivo.However, a recently completed phase-II clinical trial with brain-tumorpatients yielded mixed outcomes; while there was some improvedtherapeutic activity when O⁶-BG and temozolomide were given to patientswith temozolomide-resistant anaplastic glioma, there seemed to be nosignificant restoration of temozolomide sensitivity in patients withtemozolomide-resistant glioblastoma multiforme.

4. COMBINATION TREATMENTS

In one embodiment, a 2,4-ds-PBN therapy (optionally including TMZ) maybe used in conjunction with another glioma therapy, such as radiation,PCV, DFMO, CCNU or BCNU. These compositions would be provided in acombined amount effective to kill or inhibit proliferation of the cell.This process may involve contacting the cells with the agents at thesame time. This may be achieved by contacting the cell with a singlecomposition or pharmacological formulation that includes both agents, orby contacting the cell with two distinct compositions or formulations,at the same time, wherein one composition includes 2,4-ds-PBN and theother includes the second agent.

Alternatively, the 2,4-ds-PBN therapy may precede or follow the otheragent treatment by intervals ranging from minutes to weeks. Inembodiments where the other agent and 2,4-ds-PBN are applied separatelyto the cell, tissue or organism, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the agents would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one may contact the cell with both modalities withinabout 12-24 h of each other and, more preferably, within about 6-12 h ofeach other. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several d (2, 3, 4,5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between therespective administrations.

Multiple administrations of each agent are contemplated. For example,where the 2,4-ds-PBN therapy (optionally including TMZ or other AGTinhibitor) is “A” and the secondary agent or therapy is “B,” thefollowing are contemplated:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Patients will be evaluated for neurological changes considered to beindependent of tumor and graded using NCI Common Toxicity Criteria(neurotoxicity). Aside from baseline audiometric testing, repeataudiometric testing for ototoxicity is performed at the physician'sdiscretion for patients who had evidence of hearing loss or progressionof hearing loss by neurological examination. In addition, blood countsare performed biweekly, and serum creatinine, alkaline phosphatase,bilirubin and alanine amino-transferase tests are performed before eachcycle. Doses may be modified during the course of treatment, primarilybased on neutrophil and platelet counts (vincristine, lomustine andmatulane) or ototoxicity (DFMO). Occasionally, DFMO dose reductions arerequired for diarrhea.

A. PCV

PCV is a drug combination therapy employing three different agents—ahydrazine derivative, matulane, a nitrosourea, lomustine, and a tubulininteractive agent, vincristine. It has been used in a number of clinicaltrials, most notably by the inventor in assessing its effect onhigh-grade glioma and medulloblastoma tumors. The major side-effectobserved with PCV was dose-limiting myelotoxicity. Each of thecomponents of PCV is described below.

It should be noted that the present invention could include the use ofBCNU rather than of CCNU (lomustine) since both are nitrosoureas. Italso is contemplated that one could use CCNU and procarbazine or BCNUand procarbazine, without vincristine, since vincristine is usuallyconsidered to be the least active of the drugs in the PCV combination.

Both hydrazines and nitrosoureas are alkylating agents. As a group,alkylating agents form covalent chemical adducts with cellular DNA, RNAand protein molecules and with smaller amino acids, glutathione andsimilar chemicals. Generally, these alkylating agents react with anucleophilic atom in a cellular constituent, such as an amino, carboxyl,phosphate, sulfhydryl group in nucleic acids, proteins, amino acids, orglutathione. The mechanism and the role of these alkylating agents incancer therapy is not well understood. In addition to hydrazine andnitrosoureas, alkylating agents include: triazenes such as dacarabzineand temozolomide, nitrogen mustards such as chlorambucil,cyclophosphamide, isofamide, mechlorethamine, melphalan, uracil mustard;aziridine such as thiotepa; methanesulphonate esters such as busulfan;platinum complexes such as cisplatin, carboplatin; bioreductivealkylators, such as mitomycin and altretemine. Any of these compoundsmay be used together or individually, in combination with the compoundsof the present invention.

i. Hydrazine and Triazene Derivatives

Hydrazine and triazene derivatives are similar to nitrosoureas in thatthey decompose spontaneously or are metabolized to produce alkylcarbonium ions, which alkylate DNA. This class of compounds includesmatulane, dacarbazine and temozolomide.

The active ingredient in matulane is Procarbazine Hydrochloride(N-isopropl-alpha-(2-methylhydrazino)-p-toluamide monohydrochloride). Itis available from Roche Laboratories, Inc. It was approved in 1969 fortreatment of Hodgkins' Disease. The typical form is an oral capsule thatcontains 50 mg procarbazine as the hydrochloride. Dosages vary dependingupon whether procarbazine is being used as a combination drug with otheranticancer drugs or as a single therapeutic agent. A suggested guidelineper the PDR for single agent use is 100 mg two times daily for 14 days.

The exact mode of actions of matulane is not clear. There is someevidence that the drug acts by inhibition of protein, RNA and DNAsynthesis. It is primarily metabolized in the liver and kidneys andappears to be auto-oxidized to the azo derivative with the release ofhydrogen peroxide. The azo derivative isomerizes to the hydrazone and,following hydrolysis, splits into a benzylaldehyde derivative andmethylhydrazine. The methylhydrazine is further degraded to CO₂ and CH₄,and possibly hydrazine, whereas the aldehyde is oxidized to acid whichis excreted in the urine.

Matulane exhibits monamine oxidase inhibitory activity (MAOI), so a dietthat restricts foods which contain high tyramine content should befollowed. Drugs to be avoided during therapy include antihistamines,sympathomimetics, barbiturates, narcotics, hypotensive agents orphenothiazines, and ethyl alcohol. Some foods are also to be avoidedduring procarbazine such as naturally aged cheeses, chocolates, nuts,and bananas as they could theoretically lead to a hypertensivecomplication in some patients. Also, unacceptable toxicity may occur ifmatulane is used in patients with impairment of renal and/or hepaticfunction. Treatment may be curtailed in the event of central nervoussystem signs or symptoms such as paresthesias, neuropathies orconfusion; neutropenia (absolute neutrophil count under 1500/μl),thrombocytopenia (platelets under 100,000/μl), hypersensitivityreaction, ulceraction or persistent spot of soreness around the oralcavity, diarrhea or loose stools, hemorrhage or bleeding tendencies.

Adverse but expected reactions include leukopenia, neutropenia, anemia,and thrombocytopenia. Commonly reported acute side effects are nauseaand vomiting during or shortly after dose administration.

ii. Nitrosoureas

Nitrosoureas represent a group of therapeutic alklyating agents. Thisclass of compounds includes lomustine, carmustine, semustine,steptozocin, and nimustine.

(a) Lomustine

Lomustine is a synthetic alkylating agent, also known as CCNU, with thechemical name of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea. It wasapproved in 1977 for treatment of brain tumors and Hodgkin's Disease. Itis available from Bristol Myers Squibb as oral capsule, available in 10mg, 40 mg and 100 mg forms. Dosages may vary depending upon whetherlomustine is being used as a single agent or in a combination inaddition to other chemotherapeutic agents. As a single agent inpreviously untreated patients, the recommended dosages per the PDR is130 mg as a single oral dose every 6 weeks. Lomustine crosses the bloodbrain barrier.

It is believed that CCNU alkylates DNA and RNA. It is cross-resistantwith other nitrosoureas and some but not all alkylating agents. It mayalso inhibit several key enzymatic processes by carbamoylation of aminoacids in proteins.

The most common and severe toxic side effects are bone marrowsuppression leading to thrombocytopenia and leukopenia, which maycontribute to bleeding and infections. Bone marrow toxicity iscumulative and thus dosage adjustments must be considered on the basisof the nadir blood counts from prior doses.

(b) Carmustine

Carmustine, also known as BCNU, with the chemical name ofN,N′-Bis(2-chloroethyl)-N-nitrosurea, is a nitrosurea alkylating agentapproved by the FDA in 1977. Carmustine has been used for many years fortreatment of primary brain tumors and is used for the treatment ofgliomas. Carmustine is available from Bristol Meyers Squibb in packagescontaining vials of 10 mg carmustine and 3 ml sterile diluent fordelivered by i.v. injection. As a single agent carmustine isadministered at about 150-200 mg/m² every 6 weeks. In combinationregimens, carmustine may be given in does similar to those of lomustine.An alternative mode of delivery is by wafers implanted directly into thetumor site (Gliadel® Wafer).

Potential side effects include bone marrow suppression, anemia,diarrhea, low white blood cell and platelet counts, pulmonary toxicityand swallowing difficulties.

iii. Tubulin Interactive Agents

Tubulin interactive agents interfere with cell division by binding tospecific sites on Tubulin, a protein that polymerizes to form cellularmicrotubules. Microtubules are critical cell structure units. When theinteractive agents bind on the protein, the cell cannot properly formmicrotubules. Tubulin interactive agents include vincristine andvinblastine, both alkaloids and the taxanes, such as paclitaxel anddocetaxel.

Vincristine, is available as Oncovin™ from Eli Lilly & Company and asVincristine Sulfate from Faulding. Also called vincaleukoblastine, a22-oxo-, sulfate (1:1) (salt), the salt of an alkaloid obtained from acommon flowering herb, the periwinkle plant. It is delivered byintravenous injection. It was approved in 1963 on label for Ewing'sSarcoma, rhabdomyosarcoma, Wilm's Tumor, neuroblastoma, Hodgkin'sDisease and leukemia.

The mechanism of action remains under investigation; however, there isan indication that inhibition of microtobule formation in the mitoticspindle, resulting in an arrest of dividing cells at the metaphasestate, is involved. The liver is the major excretory organ. Most of anintravenous dose of Vincristine is excreted into the bile after rapidtissue binding. Vincristine does not appear to cross the blood brainbarrier.

Vincristine has been reported to reduce blood levels of antiseizuremedications and to increase seizure activity. The most common adversereaction is hair loss. Leukopenia, neuritic pain and constipation occur,but usually for less than 7 days.

B. DFMO

Numerous highly proliferative types of cancer are associated withincreased levels of the polyamines putrescine, spermidine, and sperminein tumor tissue and blood and urine of mammals with cancer. Studies haveshown that this can be related to increased polyamine synthesis by therate-limiting enzyme, ornithine decarboxylase (ODC). The pathway forpolyamine synthesis begins with L-ornithine. This natural amino acid,although not normally incorporated into proteins, is part of the ureacycle which metabolizes arginine to ornithine and urea. Ornithine isconverted by ornithine decarboxylase (ODC) to putrescine and CO₂ and isconsidered to be the rate-limiting step in the production of polyamines.With the addition of propylamine donated from S-adenosylmethionine,putrescine is converted to spermidine. Spermidine is then converted tospermine by spermine synthetase, again in association with thedecarboxylation of S-adenosylmethionine. Putrescine, spermidine andspermine represent the three major polyamines in mammalian tissues.Polyamines are found in animal tissues and microorganisms and are knownto play an important role in cell growth and proliferation. Although theexact mechanism of the role of the polyamines in cell growth andproliferation is not known, it appears that the polyamines mayfacilitate macromolecular processes such as DNA, RNA, or proteinsynthesis. Polyamine levels are known to be high in the testes, ventralprostate, and thymus, in psoriatic skin lesions, and in other cellsundergoing rapid growth processes.

It also is well known that the rapid proliferation of tumor tissue ismarked by an abnormal elevation of polyamine levels. Hence, thepolyamines also may play an important role in the maintenance of tumorgrowth. Thus, ODC inhibitors, such as DFMO, may exert their therapeuticeffect by blocking the formation of the polyamines and thereby slowing,interrupting, or arresting the proliferation and metastases of the tumortissue.

DFMO (α-difluoromethylornithine, eflornithine, Ornidyl®) is a structuralanalog of the amino acid L-ornithine and has a chemical formulaC₆H₁₂N₂O₂F₂. DFMO can be employed in the methods of the invention as aracemic (50/50) mixture of D- and L-enantiomers, or as a mixture of D-and L-isomers where the D-isomer is enriched relative to the L-isomer,for example, 70%, 80%, 90% or more by weight of the D-isomer relative tothe L-isomer. The DFMO employed may also be substantially free of theL-enantiomer.

The dose limiting toxic effect of DFMO is thrombocytopenia (abnormallyfew platelets in the blood), which occurs in about 50% of patients,leukopenia (abnormally few leukocytes), or anemia. This toxic effect isrelatively harmless and reversible and cease upon withdrawal of thedrug.

The effect of an ODC inhibitor for the control of the growth rate ofrapidly proliferating tumor tissue has been assessed in standard animaltumor models. For example, the anti-tumor effect of DFMO has beendemonstrated in the following animal tumor models: L1210 leukemia inmice, EMT6 tumor in Balb/C mice, 7,12-dimethylbenzanthracene-induced(DMBA-induced) mammary tumor in rats, and DFMO Morris 7288C or 5123hepatoma in Buffalo rats. In addition, the anti-tumor effect of DFMO incombination with various cytotoxic agents has been demonstrated asfollows: (a) in combination with vindesine or adriamycin in L1210leukemia in mice, in Morris 7288C hepatoma in Buffalo rats, and in EMT6tumor in mice, (b) in combination with cytosine arabinoside in L1210leukemia in mice, (c) in combination with methotrexate in L1210 leukemiain mice, (d) in combination with cyclophosphamide in EMT6 tumor in miceand in DMBA-induced tumor in mice, (e) in combination with BCNU in mouseglioma 26 brain tumor, and (f) in combination with MGBG in L1210leukemia in mice, in Morris 7288C hepatoma in Buffalo rats, in P388lymphocytic leukemia in mice, and in S-180 sarcoma in mice.

Although DFMO can effectively block tumor putrescine biosynthesis, theresultant antitumor effect is cytostasis, not cytotoxicity. For example,DFMO reduces the growth rate of an MCA sarcoma, but does not producetumor regression. This finding is consistent with reports of otherinvestigators who showed that DFMO is a cytostatic agent. However,studies indicate that a significant role may exist for DFMO agents,permitting the future development of combination chemotherapeuticregimens which incorporate DFMO.

The initial promise of DFMO as a therapeutic ODC inhibitor for use inthe treatment of various neoplasias has dimmed somewhat because,although DFMO does, in fact, irreversibly inhibit ODC activity, cellstreated in vivo with DFMO significantly increase their uptake ofexogenous putrescine as described in U.S. Pat. No. 4,925,835. Theintercellular transport mechanisms of the cell do an “end run” aroundthe DFMO-impaired ODC activity by importing putrescine from theextracellular milieu. Therefore, DFMO's effect in vivo is far poorerthan in vitro. So, while DFMO treatment effectively inhibitsintracellular putrescine neogenesis, it also results in increased uptakeof extracellular putrescine, thereby offsetting its ODC inhibitoryeffect.

This problem is compounded by the fact that putrescine is present inmany common foods, such as grapefruit juice, which containsapproximately 400 ppm putrescine. This makes it virtually impossible toprovide a patient a nutritionally sufficient diet which is free ofputrescine. Therefore, DFMO-treated cells are capable of importingsufficient amounts of extracellular putrescine to support cell division.

Strategies to make DFMO more acceptable to human patients are describedin U.S. Pat. No. 4,859,452 (incorporated by reference). Formulations ofDFMO are described which include essential amino acids in combinationwith either arginine or ornithine to help reduce DFMO-inducedtoxicities.

C. O6-Alkyguanine-DNA Alkyltransferase Inhibitors

O6-alkylguanina-DNA alkyltransferase (AGT) is a target for inhibitorydrugs such as temozolomide. Other AGT inhibitors may also prove usefulin accordance with the disclosed methods.

D. Radiation

Factors that cause DNA damage and have been used extensively for cancertherapy and include what are commonly known as γ-rays, X-rays, and/orthe directed delivery of radioisotopes to tumor cells. Other forms ofDNA damaging factors are also contemplated such as microwaves andUV-irradiation. It is most likely that all of these factors effect abroad range of damage on DNA, on the precursors of DNA, on thereplication and repair of DNA, and on the assembly and maintenance ofchromosomes. Dosage ranges for X-rays range from daily doses of 50 to200 roentgens for prolonged periods of time (3 to 4 wk), to single dosesof 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely,and depend on the half-life of the isotope, the strength and type ofradiation emitted, and the uptake by the neoplastic cells. The terms“contacted” and “exposed,” when applied to a cell, are used herein todescribe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

E. Surgery

Approximately 60% of persons with cancer will undergo surgery of sometype, which includes preventative, diagnostic or staging, curative andpalliative surgery. Curative surgery as a cancer treatment may be usedin conjunction with other therapies, such as the treatment of thepresent invention, chemotherapy, radiotherapy, hormonal therapy, genetherapy, immunotherapy and/or alternative therapies. Curative surgeryincludes resection in which all or part of cancerous tissue isphysically removed, excised, and/or destroyed. Tumor resection refers tophysical removal of at least part of a tumor. In addition to tumorresection, treatment by surgery includes laser surgery, cryosurgery,electrosurgery, and microscopically controlled surgery (Mohs' surgery).It is further contemplated that the present invention may be used inconjunction with removal of superficial cancers, precancers, orincidental amounts of normal tissue.

5. LIPOSACCHARIDE BINDING PROTEIN

A. LBP

Lipopolysaccharide binding protein is a protein that in humans isencoded by the LBP gene. LBP is a soluble acute-phase protein that bindsto bacterial lipopolysaccharide (or LPS) to elicit immune responses bypresenting the LPS to important cell surface pattern recognitionreceptors called CD14 and TLR4. The protein encoded by this gene isinvolved in the acute-phase immunologic response to gram-negativebacterial infections. Gram-negative bacteria contain a glycolipid,lipopolysaccharide (LPS), on their outer cell wall. Together withbactericidal permeability-increasing protein (BPI), the encoded proteinbinds LPS and interacts with the CD14 receptor, probably playing a rolein regulating LPS-dependent monocyte responses. Studies in mice suggestthat the encoded protein is necessary for the rapid acute-phase responseto LPS but not for the clearance of LPS from circulation. This proteinis part of a family of structurally and functionally related proteins,including BPI, plasma cholesteryl ester transfer protein (CETP), andphospholipid transfer protein (PLTP). This gene is found on chromosome20, immediately downstream of the BPI gene. Lipopolysaccharide-bindingprotein has been shown to interact with CD14, TLR2, TLR4 and theco-receptor MD-2.

B. LBP Conjugates and Imaging

As first reported here by the inventors, LBP levels are elevated inhigh-grade human patient gliomas compared with tumors classified aslow-grade gliomas. Thus, LBP conjugates for use in accordance withdiagnostic methods disclosed herein include diagnostic conjugates of LBPlinked to fluorescent labels, radioactive labels and contrast agents.These conjugates can be administered to a subject and their presence inthe body, and in particular at glioma sites, can be determined usinghand-held fluorescence scanners, near infrared scanners, MRI devices,and PET scanners. This approach can also be used to monitor efficacy oftreatments, with reductions in LBP levels indicating therapeuticbenefit.

6. PHARMACEUTICAL FORMULATIONS

The present invention discloses numerous compositions, which in certainaspects of the invention, are administered to animals. For example,2,4-ds-PBN, as well as various secondary chemotherapeutic agents, willbe formulated for administration. Where clinical applications arecontemplated, it will be necessary to prepare pharmaceuticalcompositions of these compounds and compositions in a form appropriatefor the intended application. Generally, this will entail preparingcompositions that are essentially free of pyrogens, as well as otherimpurities that could be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender agents suitable for introduction into a patient. Aqueouscompositions of the present invention comprise an effective amount ofthe agent, dissolved or dispersed in a pharmaceutically acceptablecarrier or aqueous medium. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients, such as other anti-cancer agents, can also be incorporatedinto the compositions.

Solutions of the active ingredients as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed withsurfactant, such as hydroxypropylcellulose. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, mixtures thereof andin oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent growth of microorganisms.Intravenous vehicles include fluid and nutrient replenishers.Preservatives include antimicrobial agents, anti-oxidants, chelatingagents and inert gases. The pH and exact concentration of the variouscomponents in the pharmaceutical are adjusted according to well-knownparameters.

An effective amount of the agents is determined based on the intendedgoal. The term “unit dose” refers to a physically discrete unit suitablefor use in a subject, each unit containing a predetermined quantity ofthe therapeutic composition calculated to produce the desired responsein association with its administration, i.e., the appropriate route andtreatment regimen. The quantity to be administered, both according tonumber of treatments and unit dose, depends on the subject to betreated, the state of the subject, and the protection desired. Preciseamounts of the therapeutic composition also depend on the judgment ofthe practitioner and are peculiar to each individual.

A. Enteral Administration

The active compounds of the present invention can advantageously beformulated for enteral administration, e.g., formulated for oraladministration. The pharmaceutical forms may include sesame oil, peanutoil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of ingestible compositions, including tables,pills and capsules. Also, it is contemplated that the agents of thepresent invention can be provided in the form of a food additive andincorporated into a daily dietary program. All of these forms aregenerally selected to be sterile and stable under the conditions ofmanufacture and storage.

The active compounds may be formulated into a composition in a neutralor salt form. Pharmaceutically acceptable salts, include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial ad antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the particular methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

B. Other Routes of Administration

In addition to the compounds formulated for enteral administration,parenteral formulations such as intravenous or intramuscular injectionare envisioned. Administration may also be nasal, buccal, rectal,vaginal or topical. Alternatively, administration may be by intradermal,subcutaneous, or intraperitoneal injection. Also contemplated iscontinuous perfusion via catheter. Such compositions would normally beadministered as pharmaceutically acceptable compositions, describedsupra.

7. EXAMPLES

The following examples are included to demonstrate particularembodiments of the invention. It should be appreciated by those of skillin the art that the techniques disclosed in the examples which followrepresent techniques discovered by the inventor to function well in thepractice of the invention, and thus can be considered to constitutespecifically contemplated modes for its practice. However, those ofskill in the art should, in light of the present disclosure, appreciatethat many changes can be made in the specific embodiments which aredisclosed and still obtain a like or similar result without departingfrom the spirit and scope of the invention.

Example 1 Introduction and Study Design

Orthotopic G55 human GBM xenografts in nude mice is an established GBMmodel that the inventor has used in previous publications, and continuesto use in his group. This model emulates a very aggressive GBM. For thismodel, the inventor had previous data for animal survival, tumor volumeand vascular perfusion data for untreated animals, as well asTMZ-treated animals. He also had preliminary data that indicates thatG55 cells are TMZ-resistant, and therefore this finding further supportsthe use of these cells as an ideal xenograft model for TMZ-resistance.

The inventor conducted a study with four treatment groups. For twogroups, the inventor required a minimum of 5 animals per group. Thesewere the OKN alone, and OKN+TMZ groups. For the other two groups(untreated and TMZ alone), the inventor included preexisting data tomake up a group of 5 for each, and he included 2 untreated and 2 TMZ inthe study conducted.

The inventor used MRI to obtain tumor volumes that were used to assesstreatment response. He also obtained vascular perfusion data to assessthe effects of treatment on tumor vascularity. Tumor vascular perfusionrates (measured as relative cerebral blood flow or rCBF) are calculatedfrom the perfusion images. Tumor vascular perfusion rates are decreasedin untreated tumors due to a disorganized vasculature due toangiogenesis. From normalized differences in rCBF, this would result ina substantial change in rcBF. Restoration of the tumor vascularperfusion rates via anti-cancer treatment can be indicative of ananti-angiogenesis effect. From normalized changes in rCBF, this wouldresult in a relatively small difference in rCBF if the treatment effecthad affected tumor vasculature. The inventor also assessed percentanimal survival. For the MRI study, he initially indicated that aminimum of 3 time points was needed for the untreated group, and atleast 5 time-points for the treated animals to assess tumor growth andvascular changes with MRI, whereas in actuality the inventor obtained atleast 5 time-points for the untreated mice and well over 10 time-pointsfor the treated mice, due to a positive treatment response.

The inventor also discovered a potential biomarker, LBP(lipopolysaccharide binding protein), that is elevated in tumors of ratF98 glioma-bearing rats (assessed by ELISA), and in GBM human tissuesamples (assessed by immunohistochemistry). This biomarker wasdiscovered from microarray data from the rat F98 glioma model whereuntreated tumors had high levels of LBP, and OKN-007 treatment hada >2-fold decrease in gene expression for LBP. Also in the rat F98glioma model, OKN-007 was found to significantly decrease LBP proteinlevels (assessed by ELISA) in both tumor tissue (p<0.0001) and bloodserum (p<0.001) when compared to untreated tumor-bearing animals. Theinventor used an ELISA kit for LBP to assess this biomarker as anefficacy marker in both G55-tumor-bearing mice in both tumor tissue andblood serum, either untreated or treated with TMZ, OKN-007 or TMZ+OKN-007.

Example 2 Methods

Cell culture. G55 cells were obtained from Dr. Michael E. Sughrue (Univ.of Oklahoma Health Sciences Center). G55 cells were cultured in DMEM(LifeTechnologies, Waltham, Ma.) supplemented with 10% cosmic calf serum(CCS; HyClone, Logan, Utah) and 1% penicillin/streptomycin.

Mice and Treatments. Animal studies were conducted in accordance to theOMRF IACUC (Institutional Animal Care and Use Committee) policies, whichfollow NIH guidelines. Two-month-old male nude mice (Hsd:AthymicNude-Foxnlnu mice; Harlan Inc., Indianapolis, Ind.) were implantedintra-cerebrally with human G55 xenograft cells (1×10⁷) per mL suspendedin 4 μL in cell culture media of 1% aragose solution. Once tumorsreached 10-15 mm³ (determined via MRI), mice were either treated every 3days with OKN-007 (150 mg/kg; via drinking water continuously) or TMZ(30 mg/kg via gavage every 3 days). TMZ was dissolved in 5% DMSO and 5%solutol-15 in sterile saline. Mice were treated until the tumor reached100-150 mm³ or for a total of up to 60 days after tumors were detectedby MRI (>5 mm³).

MRI. MRI experiments were performed on a Bruker Bio-spec 7.0 Tesla/30-cmhorizontal-bore magnet imaging system. Animals were immobilized by using1.5-2.5% isoflurane and 0.8 L/min O₂ and placed in a 72-mm quadraturevolume coil for signal transmission, and a surface mouse-head coil wasused for signal reception. T2-weighted imaging was obtained, and tumorvolumes were calculated from MRI datasets.

Perfusion imaging. In order to assess microvascular alterationsassociated with tumor capillaries, the perfusion imaging method,arterial spin labeling (ASL), was used. Perfusion maps were obtained ona single axial slice of the brain located on the point of therostro-caudal axis where the tumor has the largest cross-section. Fiveregions of interest (ROIs) were manually outlined around the tumor, andappropriate ROIs were also taken from the contralateral side of thebrain for comparison purposes. To calculate the differences in rCBFvalues, tumor rCBF values were obtained at late (days 18-26 followingintracerebral implantation of cells for untreated mice) and early (days10-13 following cell implantation) tumor stages, and normalized to rCBFvalues in the contralateral brain region of corresponding animals.

Lipopolysaccharide binding protein (LBP) ELISA assay. The LBP ELISA kit(Antibodies-Online.com; ABIN370808) is specific for mouse LBP levels inserum, plasma and tissue samples. This is a colorimetric assay that wasread on a micro plate reader for measurement of absorbance at 450/620nm. LBP concentrations were calculated from a standard curve andmultiplied by appropriate dilution factors.

Statistical analyses. Survival curves were analyzed using Kaplan-Meiercurves. Tumor volumes, changes in normalized rCBF, and LBP levels werecompared by two-way ANOVA with multiple comparisons. Data wererepresented as mean±SD, and P-values of either *0.05, **0.01, ***0.001,and ****0.0001 were considered statistically significant.

Example 3 Results

Animal survival (see FIG. 1 ). The percent animal survival dataindicated that 60% of the combined therapy (OKN-007+TMZ)-treated miceremained alive 60 days following tumor detection and treated for over 50days. One of the OKN-007-treated mice (20% of the mice treated) was alsoalive 60 days following tumor detection. Statistical analysis indicatedthat all of the treated mice (either OKN-007 alone, TMZ alone orcombined therapy) were found to have a significant increase in percentsurvival, when compared to untreated (UT) G55 glioma-bearing mice. Itwas also found that the combined therapy mice had a significantly longersurvival than TMZ-treated mice. There was no significance between theOKN-007-treated and combined therapy mice. If the animal group numberswere larger, perhaps a significant difference could be obtained betweenthe OKN-007-treated and combined therapy mice, as the p-value was 0.07between these two groups.

Tumor volumes (see FIGS. 2-4 ). The tumor volumes were compared at thesame time-period when the UT mice were euthanized, i.e., tumor volumesreached 150 mm³ or larger (at 19-22 days following tumor detection), aswell as at the last time-point for each treatment group. At 19-22 daysfollowing tumor detection, mice that were treated with either TMZ,OKN-007 or combined therapy, were all found to have significantlydecreased tumor volumes when compared to UT mice (FIG. 2 ). None of thetreated mice were found to significantly differ from each other in tumorvolumes, however the combined therapy had the lowest tumor volume meanwhen compared to either TMZ- or OKN-007-treated mice. As the samplesizes were small (n=5), there may be a significant difference betweentreatment groups if the animal numbers were increased. The TMZ group hada large variance, which the inventor has also observed in two otherstudies that he have conducted. Representative MR images depictingtumors (mid-tumor regions) are also shown for each treatment groupinvestigated (FIG. 3 ). At the last time-point for each treatment group,the tumor volumes for the TMZ and combined therapy groups were found tobe significantly smaller when compared to UT mice (FIG. 4 ). Due to thelarge variance in the OKN-007-treated group, there was no significantdifference in tumor volumes when compared to UT mice, or any of theother treated groups (TMZ alone, or combined therapy). Tumor volumesobtained at multiple time-points are depicted in the graph below (FIG.5A). A treatment window is depicted, where tumors are treated whentumors reach≥10 mm³.

Tumor vasculature perfusion rates (normalized differences in rCBF) (seeFIG. 5B). Normalized differences in tumor rCBF were found tosignificantly decrease in all treated mice, compared to UT mice. Therewere no significant differences between treated groups, due to the smallnumber of animals per group, however both the combined therapy andOKN-007-treated groups seemed to have more normalized perfusion rates intheir tumors, compared to TMZ treatment. Representative morphological MRimages and their corresponding perfusion maps are shown for eachtreatment group.

LBP ELISA assay (see FIGS. 6 and 7 ). The LBP levels in theOKN-007-treated or combined therapy mice were significantly less intumor tissues, compared to UT mice (FIG. 6 ). In tumor tissues, theTMZ-treated group LBP levels were not found to be significantlydifferent to UT mouse tumors. There was no significant differencebetween the OKN-007-treated group from the combined therapy in tumor LBPlevels. The effect could have been mainly contributed by the effect ofOKN-007. The serum LBP levels were found to be significantly less in alltreatment groups compared to UT mouse samples, indicating a generaltreatment response (FIG. 7 ).

In vitro IC₅₀ assessment of TMZ-resistant and TMZ-sensitive GBM celllines combined with OKN-007 (FIG. 8 ). OKN-007 has been previously shownby the inventors that it has anti-tumor activity in glioblastoma (GBM)pre-clinical models. In vitro data demonstrate that OKN-007 can decreaseTMZ (temozolomide)-resistant GBM cell lines (T98G and G55), when theyare combined together.

Liposaccharide binding protein (LBP). During a detailed study assessingthe mechanism of OKN-007 action, a transcriptional microarray was usedto elucidate specific genetic alterations associated with OKN-007treatment in a rat F98 glioma model. LBP was down-regulated inOKN-treated rat F98 gliomas (compared to untreated), and is associatedwith TGF-β1. LPB (lipopolysaccharide-binding protein), which wasdiscovered 27 years ago and named after the ability to bind LPS, isneeded to combat infections, and is involved in innate and adaptiveimmunity. The main mechanism of action of LBP is still not clear.

OKN-007 effects on LPB (FIGS. 9 and 10 ). Levels of protein LBP in tumortissues of untreated (Tumor) and OKN-007-treated (OKN-Treated Tumor) F98glioma bearing rats are shown in FIG. 9 . Normal brain tissues wereobtained from untreated (Control) and OKN-007-treated (Control+OKN)rats. There was a significant decrease in LBP levels in OKN-007-treatedgliomas compared to untreated (p<0.0001). Control tissues (untreated ortreated) were also significantly lower (p<0.0001)

Levels of protein LBP in serum of untreated (Tumor) and OKN-007- treated(OKN-Treated) F98 glioma rats are shown in FIG. 10 . Serum from normalrats (Control) was also obtained. Serum levels of LBP were significantlylower in OKN-treated F98 glioma-bearing rats compared to untreatedtumor-bearing rats (p<0.001), in normal rats vs. untreated tumor-bearing(p<0.0001), or normal rats vs. OKN-007-treated glioma-bearing rats(p<0.05).

Immunohistochemistry levels of LBP in a high grade glioma (FIG. 11A),compared to low grade glioma (FIG. 11B), indicate a significant increasein LBP in high grade gliomas (FIG. 11C). LBP was found to besignificantly lowered by OKN-007 or by combined OKN-007+TMZ treatment inG55 human GBMV xenografts in nude mice in both tumor tissues (leftgraph) and blood serum (right graph), compared to untreated tumors. FIG.12 .

Conclusions. The combined therapy (OKN-007+TMZ) seemed to have anoverall favorable response in increased animal survival, decreased tumorvolumes, decreased change in rCBF, and decreased levels of LBP in bothtumor tissue and blood serum. It is possible that an increase in animalnumbers would show significant differences in animal survival, tumorvolumes and changes in rCBF when comparing the 3 treatment groups (TMZalone, OKN-007 alone and combined therapy). It would also beadvantageous to conduct the study in another TMZ-resistant model (eitherxenograft model of another TMZ-resistant GBM cell line, or apatient-derived xenograft model using GBM cells from a patient thatfailed TMZ treatment). In conclusion, the data supports that combinedOKN-007 and TMZ treatment indicates an overall improvement compared toeither TMZ or OKN-007 sole treatments and should be considered forclinical investigation. However, in order to convince other cancerresearchers regarding a publication, an increase in animal numbers, andperhaps using another xenograft model, would strengthen the overalloutcome.

Example 4 Materials and Methods In Vivo Studies

Mice and Treatments. Animal studies were conducted in accordance to theOMRF IACUC (Institutional Animal Care and Use Committee) policies, whichfollow NIH guidelines. For the F98 rat glioma cell implantation model,F98 cells (10⁵ in 10-μ1 volume) were intracerebrally implanted with astereotaxic device (2 mm lateral and 2 mm anterior to the bregma, and ata 3 mm depth) in a total of 15 Fischer 344 rats (male 200-250 gm). Theanimals were divided into two groups once tumors reached 10-20 mm³ involume (as determined by MRI): OKN-007 treated (n=8) and untreated (UT)(n=7) groups. Rats were treated until tumors reached 200-250 mm³ involume or for a total of 4-6 weeks. For the G55 GBM cell implantationmodel, two-month-old male nude mice (Hsd:Athymic Nude-Foxnlnu mice;Harlan Inc., Indianapolis, IN) were implanted intracerebrally with humanG55 xenograft cells (1×10⁶) per mL suspended in 4 μL in cell culturemedia of 1% agarose solution. Once tumors reached 10-15 mm³ (determinedvia MRI), mice were either treated daily with OKN-007 in the drinkingwater (150 mg/kg; 0.20% w/v for a 20 g mouse), or every 3 days with TMZ(30 mg/kg). OKN-007 was dissolved in water and made fresh every 2 days.Water bottles were weighed, and the amount of OKN-007 consumed per mousewas determined. No significant deviation was observed in the volume ofliquid uptake of OKN-007 in these mice. The average intake of OKN-007was approximately 140-150 mg/kg/day/mouse. TMZ was dissolved in 5% DMSOand 5% solutol-15 in sterile saline and administered via gavage. Micewere treated until the tumors reached 100-150 mm³ or for a total of 4-6weeks. All groups were stratified to ensure that tumor sizes weresimilar before initiation of treatment.

Magnetic resonance imaging (MRI). MRI experiments were performed on aBruker Bio-spec 7.0 Tesla/30-cm horizontal-bore magnet imaging system.Animals were immobilized by using 1.5-2.5% isoflurane and 0.8 L/min O₂and placed in a 72-mm quadrature volume coil for signal transmission,and either a surface rat-head or mouse-head coil were used for signalreception. T2-weighted morphological imaging was obtained with a slicethickness of 0.5 mm, a FOV of 4×5 cm² for rats or 2×2 cm² for mice, withan approximate in-plane resolution of 150 μm for rats and 80 μm formice, with a repetition time (TR) of 3000 ms and an echo time (TE) of 63ms, for a total acquisition time of 13 min. Tumor volumes werecalculated from 3D MRI slices rendered MRI datasets, using Amira v5.6.0(FEI) (Zhao et al., 2018; Tang et al., 2011; Tang et al., 2016).

Perfusion imaging. In order to assess microvascular alterationsassociated with tumor capillaries, the perfusion imaging method,arterial spin labeling (ASL), was used as previously described (Ziegleret al., 2017). Perfusion maps were obtained on a single axial slice ofthe brain located on the point of the rostro-caudal axis where the tumorhad the largest cross-section. Five regions of interest (ROIs) weremanually outlined around the tumor, and appropriate ROIs were also takenfrom the contralateral side of the brain for comparison purposes. Tocalculate the differences in rCBF values, tumor rCBF values wereobtained at late (days 18-26 following intracerebral implantation ofcells for untreated mice) and early (days 10-13 following cellimplantation) tumor stages and normalized to rCBF values in thecontralateral brain region of corresponding animals. Tumor volumes weretransposed from morphological image data sets.

RNA isolation and preparation. For the rat F98 glioma study, all ratswere euthanized after the last MRI examination. The brain of each animalwas removed, and snap frozen in liquid nitrogen, before storage in a−80° C. freezer. Total RNA from all tumor tissues from all treatmentgroups was purified with an RNeasy Mini Kit (Qiagen), and quantified byspectrophotometry (Nanodrop). cDNA was synthesized using SuperScript IVReverse Transcriptase Kit (Invitrogen).

Microarray analysis. The Illumina TotalPrepTM RNA Amplification Kit wasused for labeling cRNA (Ambion, Austin, Tex.), as previously described(Griffins et al., 2009). Four×four treated/untreated samples wereprofiled using Affymetrix RaGene-1_0-st-v1 microarrays.Exon-level-summarized measures were quantile-normalized and tested fordifferential expression using Significance Analysis of Microarrays (SAM,[PMID: 11309499]) at a false discovery rate (FDR) <40% and fold change<1.5. Functional enrichment analysis was performed using IngenuityPathways Analysis (IPA) (Ingenuity® Systems, world-wide-web atingenuity.com).

Histology and immunohistochemistry (IHC). All mice were euthanized afterthe last MRI examination. Perfusion fixation (10% neutral bufferedformalin administered via a tail-vein injection) was used onanesthetized (Isoflurane) mice, and whole brain of each animal wasremoved, further preserved in 10% neutral buffered formalin, andprocessed routinely. Paraffin-embedded tissues were sectioned in 5 μmsections, mounted on super frost plus® glass slides, stained withhematoxylin and eosin (H&E), and examined by light microscopy.Immunohistochemistry (IHC) was done to establish TGFβ1 levels bystaining tissue samples with anti-TGFβ1 antibody (rabbit anti-TGFβ1,cat. No. 250876, 1 mg/mL, ABBIOTEC, San Diego, Calif.). For TGFβ1 IHC,sections were incubated in an antigen retrieval solution (citratebuffer, pH 6, Vector Laboratories, Burlingame Calif.) for 20 min in arice steamer, followed by a 20 min cool down in deionized water.

Statistical Analysis. Survival curves were analyzed using Kaplan-Meiercurves. Tumor volumes, changes in normalized rCBF, and tumor bloodvolumes were analyzed and compared by two-way ANOVA with multiplecomparisons. Data were represented as mean±SD, and P-values of either*0.05, **0.01, ***0.001, and ****0.0001 were considered statisticallysignificant. For microarray data, random variance t-statistics for eachgene was used (Wright and Simon, 2003).

In Vitro Studies

Cells and culture media. Most GBM cells were obtained from the AmericanTissue

Culture Collection (ATCC; Manassas, VA, USA) (U-138—ATCC (CRL-HTB-16)glioblastoma; LN-18—ATCC (CRL-2610) glioblastoma; LN-229—ATCC (CRL-2611)glioblastoma; and T-98G—ATCC—(CRL-1690). U-251 GBM cells were obtainedfrom Sigma-Aldrich (N#09063001 also known before as U-373 MG (ATCC®HTB-17)). G55 cells were obtained from Dr. Michael Sughrue, who was aresident at Univ. of California San Francisco (originally obtained fromC. David James (Department of Neurological Surgery, UCSF, CA, USA), whocharacterized the cells).

Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GibcoBRL, Crand Island, N.Y., USA) supplemented with 10% fetal bovine serum(Gibco) in a standard humidified incubator at 37° C. under 5% CO₂.

IC₅₀ concentrations—Protocol for TMZ sensitivity determination. Thesensitivities of the six glioma cell lines to TMZ were evaluated fromthe concentrations required for 50% growth inhibition (IC₅₀; also knownas GI₅₀) in comparison with untreated controls (Wang et al., 2017).Briefly, cells were plated at 1×10⁴ cells per well in 24-well,flat-bottomed plates and incubated with medium for 24 hrs. The cellswere subsequently washed twice with medium and incubated further withfresh medium (control) or medium containing 0.1-1,000 μM of TMZ. Pereach plate contained growth medium with +/−TMZ there was a plate withmedium containing 1 mM OKN. After exposure to the various concentrationsof TMZ for 72 hrs, cells were detached by trypsinization and the numberscounted. The experiments were repeated at least 4 times at eachconcentration.

RNA preparation. To avoid contributions from artificial sources in theexperimentally measured expression patterns, each cell line was grown infour independent cultures, and the entire process was carried outindependently on mRNA extracted from each culture.

Cell lines LN-18 and LN-229 were subjected to an assessment of theirgene expression profile for 4 groups: cells, cells with TMZ, cells withTMZ-OKN combined treatment, cells with OKN. The extracted total RNA waspurified with an RNeasy Mini Kit (Qiagen), quantified byspectrophotometry (Nanodrop).

Quantification of mRNA by real-time quantitative RT-PCR for HIF-1a, MPGand MGMT. Total RNA from all cell lines with all treatments was purifiedwith an RNeasy Mini Kit (Qiagen), and quantified by spectrophotometry(Nanodrop). cDNA was synthesized using SuperScript IV ReverseTranscriptase Kit (Invitrogen).

Target gene mRNA was amplified and measured by Bio-Rad CFX96™ Real TimeSystem. Gene expression was determined using the SYBR Select Master Mix(Applied Biosystems). Fluorescence signals, which are proportional tothe concentration of the PCR product, are measured at the end of eachcycle and immediately displayed on a computer screen, permittingreal-time monitoring of the PCR. The reaction is characterized by thepoint during cycling when amplification of PCR products is firstdetected, rather than the amount of PCR product accumulated after afixed number of cycles. The higher the starting quantity of thetemplate, the earlier a significant increase in fluorescence isobserved. The threshold cycle is defined as the fractional cycle numberat which fluorescence passes a fixed threshold above the baseline.Fluorescence data were converted into cycle threshold measurementsexported to Microsoft Excel. Glyceraldehyde-3-phosphatase dehydrogenase(GAPDH) mRNA expression levels were used as the quantitative internalcontrol. For precise quantification, the mRNA expression level of eachsample was normalized using the expression of the GAPDH gene.

All primers were synthesized by Integrated DNA Technologies. The primersused were:

HIF-1α: (SEQ ID NO: 1) F 5′-GTCGGACAGCCTCACCAAACAGAGC-3′ (SEQ ID NO: 2)R 5′-GTTAACTTGATCCAAAGCTCTGAG-3′. For MGMT: (SEQ ID NO: 3)F 5′-GGGTCTGCACGAAATAAAGC-3′, (SEQ ID NO: 4)R 5′-CTCCGGACCTCCGAGAAC-3′ [6], (SEQ ID NO: 5)5′-GTC CTA GTC CGG CGA CTT CC-3′, and (SEQ ID NO: 6)5′-CTT GTC TGG GCA GGC CCT TTG C-3′were used to amplify 603-bp transcripts of MPG [9].

ELISA for HIF-1a, MPG and MGMT. Protein expressions were assessed in allsix glioma cell lines for all four groups of treatment (cell alone,cells with TMZ, cells with OKN, cells with combined OKN and TMZ). Cellswere lysed before assaying. In brief, cells were washed by cold PBSgently, and then detached with trypsin, and collected by centrifugationat 1,000×g for 5 minutes. Then, cells were washed three times in coldPBS, then suspended in fresh lysis buffer. Lysates were centrifuged at1,500×g for 10 minutes at 2-8° C. to remove cellular debris.

The assay is based on the sandwich ELISA principle. Each well of thesupplied microtiter plate has been pre-coated with a target specificcapture antibody. Standards or samples are added to the wells and thetarget antigen binds to the capture antibody. Unbound Standard or sampleis washed away. A biotin-conjugated detection antibody is then addedwhich binds to the captured antigen. Unbound detection antibody iswashed away. An Avidin-Horseradish Peroxidase (HRP) conjugate is thenadded which binds to the biotin. Unbound Avidin-HRP conjugate is washedaway. A TMB substrate is then added which reacts with the HRP enzymeresulting in color development. A sulfuric acid stop solution is addedto terminate color development reaction and then the optical density(OD) of the well is measured at a wavelength of 450 nm±2 nm. The OD ofan unknown sample can then be compared to an OD standard curve generatedusing known antigen concentrations in order to determine its antigenconcentration.

The antigen concentration determined from ELISA was then normalized tothe total protein concentration of each cell lysate in order to havecomparison between groups. ELISA Kits for N-Methylpurine DNA Glycosylase(MPG) and for Hypoxia Inducible Factor 1 Alpha (HIF1a) were purchasedfrom CLOUD-CLONE CORP.(CCC) and for Human MGMT from LifeSpanBiosciences, Inc.

RNA-seq. Prior to RNA-seq analysis quality control measures wereimplemented. Concentration of RNA was ascertained via fluorometricanalysis on a Thermo Fisher Qubit fluorometer. Overall quality of RNAwas verified using an Agilent Tapestation instrument. Following initialQC steps sequencing libraries were generated using the Lexogen QuantseqFWD library prep kit according to the manufacturers protocol. Briefly,the first strand of cDNA was generated using 5′-tagged poly-T oligomerprimers. Following RNase digestion, the second strand of cDNA wasgenerated using 5′-tagged random primers. A subsequent PCR step withadditional primers added the complete adapter sequence to the initial 5′tags, added unique indices for demultiplexing of samples, and amplifiedthe library. Final libraries for each sample were assayed on the AgilentTapestation for appropriate size and quantity. These libraries were thenpooled in equimolar amounts as ascertained via fluorometric analyses.Final pools were absolutely quantified using qPCR on a Roche LightCycler480 instrument with Kapa Biosystems Illumina Library Quantificationreagents. Sequencing was performed on an Illumina Nextseq 500 instrumentwith High Output chemistry and 75 bp single-ended reads.

Raw sequencing files were processed with bbduk (decontamination usingKmers) (Bushnell, 2014) trimming of poly A tails and adaptor sequences.Fastqc (Andrews, 2010) and multiQC (Ewels et al., 2016) were used tocheck the quality of the resulting fastq files. High-quality scores(phred scores) of 33-36 were present in all samples with 9.3+/−1.3million reads each. Sorted bam files aligned with Tophat2 (Trapnell etal., 2009) to the GRCh38 genome were then provided to two separatepipelines in parallel, in order to analyze the robust responses fromeach package. Counts and differential gene expression were obtained withR using the ‘GenomicAlignments’ function ‘summarizeOverlaps’ and thenegative binomial generalized linear modeling package DESeq2 (Love etal., 2014).

Cell migration. For the migration study, 6-well chambers with PDMS(polydimethylsiloxane) micro-channels inside were coated with 10 μg/mlof laminin (Sigma-Aldrich) in each well. G55 cells were seeded (50×10³)in 100 μl and supplemented with 2 ml of media in each well. The chamberswere incubated in the incubator at 37° C. with 5% CO₂ until scheduledtime points. Some chambers were treated with OKN-007, TMZ or OKN-007combined with TMZ (1 μL/mL media for both OKN-007 and TMZ, as well aswhen the two are combined), and for each treatment group one well wasleft untreated as a control. Images of the cells inside themicrochannels were taken using an Olympus CK40 inverted microscope(Japan) under 10× and the distances of the same cells traveled weremeasured at 22 h, 28 h, and 46 h post seeding, cell migration speed(μm/h) was calculated. Each treatment was repeated at least 3 times anddata shown as mean ±S.D.

Statistical Analysis. RT-PCR gene and ELISA protein levels, and cellmigrations were analyzed and compared by two-way ANOVA with multiplecomparisons. RNA-seq data were analyzed using FDR<0.05 from theBenjamini-Hochberg FDR values provided by DESeq2 (Love et al., 2014;Benjamini and Hochberg, 1995; Robinson et al., 2010). Data wererepresented as mean±SD, and P-values of either *0.05, **0.01, ***0.001,and ****0.0001 were considered statistically significant.

Example 5 Results

In vivo G55 orthotopic xenograft GBM model. The percent animal survivaldata indicated that 60% of the combined therapy (OKN-007+TMZ)-treatedmice remained alive 60 days following tumor detection and treated forover 50 days (FIG. 13A). One of the OKN-007-treated mice (20% of themice treated) was also alive 60 days following tumor detection.Statistical analysis indicated that all of the treated mice (eitherOKN-007 alone, TMZ alone or combined therapy) were found to have asignificant decrease in percent survival, when compared to untreated(UT) G55 glioma-bearing mice. It was also found that the combinedtherapy mice had a significantly longer survival than TMZ-treated mice.There was no significance between the OKN-007-treated and combinedtherapy mice.

The tumor volumes were compared at the same time-period when the UT micewere euthanized, i.e., tumor volumes reached 150 mm³ or larger (at 19-22days following tumor detection), as well as at the last time-point foreach treatment group. At 19-22 days following tumor detection, mice thatwere treated with either TMZ, OKN-007 or combined therapy, were allfound to have significantly decreased tumor volumes when compared to UTmice (FIG. 13B). None of the treated mice were found to significantlydiffer from each other in tumor volumes, however the combined therapyhad the lowest tumor volume mean when compared to either TMZ- orOKN-007-treated mice. Representative MR images depicting tumors(mid-tumor regions) are also shown for each treatment group investigated(FIG. 14 ).

Normalized differences in tumor rCBF were found to significantlydecrease in all treated mice, compared to UT mice (FIG. 15E). There wereno significant differences between treated groups, due to the smallnumber of animals per group, however both the combined therapy andOKN-007-treated groups seemed to have more normalized perfusion rates intheir tumors, compared to TMZ treatment. Representative morphological MRimages (FIGS. 15 -Di) and their corresponding perfusion maps (FIGS.15A-Dii) are shown for each treatment group.

In vitro GBM cell study, From the in vitro GBM cell growth curves, itwas established from the TMZ-sensitive cells (U251, LN229) that most ofthe cells were killed more than 50% with TMZ concentrations of 100 μM orless (Table 4). For the TMZ-resistant GBM cells (T98, LN18, U138, G55),the effect of combined therapy was substantial at TMZ concentrationsless than 100 μM. The efficacy of combined therapy was significant.

TABLE 4 IC₅₀ values for human GBM cells treated with either TMZ alone(0, 0.1, 1, 10, 100 or 1000 μM) or TMZ combined with OKN (1 mM). TMZ,μM + Cell line TMZ, μM OKN-007 G55 (low) 567.4 63.4 G55 (high) 94.3 37.9U138 448.1 195.5 U251 176.5 8.0 LN229 107.5 Less 1 (<1) ~100 Less 1 (<1)LN18 773.7 31.9 T98 438.3 295.4 447.2 138.9

A concentration of 1 mM OKN was found to be just as effective inreducing cell viabilities, compared to the 2 mM concentration for somecells (LN229, U138, G55). In the other cells (U251, T98, LN18) 2 mM OKNwas found to be slightly more effective in reducing cell viabilities,compared to the 1 mM concentration.

HIF-1α, MGMT and MPG. RT-PCR (Table 5) indicated that HIF-1α gene-foldchanges had increased in all TMZ-treated and OKN-007 +TMZ-treated cells.OKN-007 increased HIF-1α gene-fold changes in all cells, except T98cells, which had decreased gene-fold changes. MGMT gene-fold change isdecreased in LN18 cells treated with either TMZ or OKN-007. OKN-007decreased MGMT gene-fold change in T98 cells. MGMT gene-fold changes areincreased in U251 cells following either TMZ, OKN-007 or combinedtreatment. MGMT gene-fold changes are increased slightly in LN18combined treatment. MPG gene-fold changes are increased in most cellsfollowing TMZ treatment, in OKN-007-treated G55 and U138 cells, and forG55, LN18 and U138 cells treated with OKN-007 combined with TMZ. Therewere decreases in MPG gene-fold changes with OKN-007-treatment for LN18,T98, U251 and LN229 cells, and also for T98 and LN229 cells treated withOKN-007 +TMZ.

TABLE 5 RT-PCR gene-fold changes for HIF-1α, MGMT and MPG inTMZ-resistant and TMZ-sensitive human GBM cells treated with either TMZ,OKN or combined OKN + TMZ, or untreated (UT). HIF-1α MGMT MPG Cells UTTMZ OKN Comb UT TMZ OKN Comb UT TMZ OKN Comb TMZ-Resistant G55 1.00 1.851.20 2.10 — — — — 1.06 2.53 4.52 3.58 LN18 1.03 2.11 1.35 2.76 1.06 0.770.42 1.26 1.00 2.95 0.60 5.40 U138 1.00 2.05 2.93 3.17 — — — — 1.13 7.1411.10 12.70 T98 1.00 1.72 0.58 1.56 1.00 1.07 0.54 1.15 1.10 1.22 0.620.73 TMZ-Sensitive U251 1.00 1.48 1.59 1.56 1.00 1.32 1.22 1.24 1.101.11 0.81 1.16 LN229 1.00 1.41 1.09 2.03 — — — — 1.10 1.22 0.62 0.73 N =2 for each cell treatment group.

ELISA established protein levels (Table 6) indicated that HIF-1a waselevated in most cells treated with TMZ or combined treatment. OKN-007decreased HIF-1a levels slightly in U138 cells, whereas in all othercells this protein was slightly elevated in this treatment group. MGMTwas elevated in G55, T98 and U251 cells treated with TMZ or combinedtreatment. OKN-007 slightly decreased MGMT in G55 and LN18 cells. MPGwas mainly only elevated in U251 cells treated with TMZ or OKN-007combined with TMZ. OKN-007 decreased MPG levels in LN229 cells.

TABLE 6 ELISA protein level (ng/mg cell lysate) changes for HIF-1α, MGMTand MPG in TMZ-resistant and TMZ-sensitive human GBM cells treated witheither TMZ, OKN or combined OKN + TMZ, or untreated (UT). HIF-1α MGMTMPG Cells UT TMZ OKN Comb UT TMZ OKN Comb UT TMZ OKN Comb TMZ-ResistantG55 0.14 1.08 0.19 1.13 0.37 1.19 0.31 1.11 1.15 1.50 1.41 1.77 LN18 076 0.83 0.92 1.07 1.23 1.28 0.98 1.31 2.18 2.27 2.38 2.20 U138 0.40 0.840.33 0.44 0.77 0.98 0.75 0.93 1.30 1.50 1.22 1 .30 T98 0.42 0.77 0.580.59 0.62 1.76 0.84 1.57 3.04 3.82 3.61 3.27 TMZ-Sensitive U251 0.301.41 0.61 0.91 0.92 1.48 0.91 1.44 2.70 4.99 2.77 4.09 LN229 0.68 0.740.66 1.14 0.67 0.75 0.69 0.89 2.34 1.93 1.88 2.23 N = 2 for each celltreatment group.

RNA-seq data. FIG. 17 shows that the LN18 cells had 37 upregulatedgenes, and 3 genes down-regulated in the TMZ+OKN group compared to TMZalone group. For sample 25 the genes seem to be all upregulated in thiscolumn. This seems to be set at a different threshold than the othercombined OKN+TMZ LN18 samples. If the threshold was reduced, then thepattern would be similar to those in columns 26 or 28 (the othercombined OKN-007+TMZ treated LN18 cells).

FIG. 16 shows that the LN18 cells had 37 upregulated genes, and 3 genesdown-regulated in the TMZ+OKN group compared to TMZ alone group. Forsample 25 the genes seem to be all upregulated in this column. Thisseems to be set at a different threshold than the other combined OKN+TMZLN18 samples. If the threshold was reduced, then the pattern would besimilar to those in columns 26 or 28 (the other combined OKN-007+TMZtreated LN18 cells). FIG. 17 shows that the LN229 cells had 21upregulated genes, and 19 genes down-regulated in the TMZ+OKN groupcompared to TMZ alone group.

In vitro cell migration study. G55 GBM cell migration studies indicatedthat the cell migration speed for OKN-treated cells was significantlydecreased at 22 h and 46 h following treatment, compared to untreated(UT) cells. Cells treated with TMZ were found to be significantlydecreased at 28 h and 46 h following treatment, compared to UT cells. Itwas also found that combined OKN+TMZ treatments significantly decreasedcell migration at 22 h and 28 h following treatment, compared to TMZalone.

Microarray data from rat F98 gliomas either UT or treated with OKN-007.Microarray analysis identified predominant downregulation of genesfollowing OKN treatment. 384 genes had at least one exon significantlydownregulated, while only 3 were upregulated (data not shown). Pathwayanalysis indicated that OKN-treated F98 tumors downregulated severalgenes associated with the extracellular matrix (ECM) (e.g., collagen andMMP genes), all with a connection to TGFβ1.

RT-PCR was able to confirm that several ECM genes were downregulatedwith OKN-treatment, compared to UT F98 tumors (Table 7).

TABLE 7 RT-PCR of RNA isolated from F98 untreated and OKN-007-treatedtumors with 2-, 5- or 10-fold changes in gene expressions. Up-regulatedDown-regulated Gene >2-fold >5-fold >10-foldGene >2-fold >5-fold >10-fold CDH4 4.06 ADAMTS1 3.57 CNTN1 15.03 CD443.03 HAPLN1 3.1 CDH3 3.70 MMP16 4.72 COL1A1 2.27 NCAM2 11.63 COL2A1 2.33SGCE 8.06 COL3A1 3.13 SPOC1 11.08 COL4A1 2.94 SYT1 19.03 COL4A2 2.63 VTN3.03 COL5A1 3.33 COL6A1 2.94 COL8A1 2.33 CTGF 2.63 ECM1 2.86 EMILIN12.27 FN1 2.63 ITGA2 3.7 ITAD 2.38 ITGB3 2.13 ITGB3 2.13 ITGB4 3.70 LAMC12.33 MMP12 16.67 MMP13 50.00 MMP14 2.86 MMP1 A 4.17 MMP2 2.86 MMP3 3.85MMP7 2.86 MMP8 3.85 MMP9 7.14 POSTN 33.33 SELE 2.86 SELL 2.13 SELP 2.13SPP1 8.33 TGFBI 2.22 THBS2 3.7 TIMP1 3.45 RGDC 3.45

Upstream regulator analysis identified TGFβ1 as the most significantinhibited upstream regulator, controlling 57 downregulated genes (FIG.20 ). TGFβ1 was itself downregulated nearly 2-fold.

Immunohistochemistry showed that TGFβ1 protein levels were substantiallyreduced in OKN-treated F98 gliomas, compared to UT tumors (FIGS. 21A-B).The decrease in TGFβ1 protein levels was also confirmed using ELISA,indicating a significantly decreased (p<0.001) level of TGFβ1 proteinexpression in OKN-treated F98 gliomas, compared to UT tumors (FIG. 21C).

Example 6 Discussion

The inventors were able to establish that OKN-007 when combined with TMZcan augment the effect of TMZ in TMZ-sensitive GBM cells, as well asrender TMZ-resistant GBM cells more sensitive to TMZ and/or the effectof OKN-007 on tumor cell growth. They were able to establish thatOKN-007, by itself, can down-regulate several genes associated with theextracellular matrix through TGF-β1, and also be effective against cellmigration. They previously found that OKN-007 can effectively inhibitcell proliferation, decrease HIF-1a and VEGFR2, which are bothassociated with angiogenesis, and increase apoptosis. When OKN-007 iscombined with TMZ, it can be effective against both TMZ-sensitive andTMZ-resistant GBM cells in vitro, and synergistically decrease tumorvolumes, as well as increase animal survival and normal vascularizationin vivo in a G55 orthotopic xenograft GBM model.

Other TMZ combined therapies that have been recently investigatedinclude: silencing GLI1, which is associated with Hedgehog signaling,and specifically affected glioma-like stem cells (U87-MG, T98G) (Melamedet al., 2018); inhibiting Wnt/β-catenin signaling, which downregulatesthe expression of aldehyde dehydrogenase isoform 3A1 (ALDH3A1) (Suwalaet al., 2018); using a miR-519a mimic, where miR-519a functions as atumor suppressor by targeting the signal transducer and activator oftranscription 3 (STAT3)-mediated autophagy, and promoting TMZ-inducedautophagy (U87-MG/TMZ) (Li et al., 2018); inhibiting PI3K to sensitizeGBM cells to TMZ (Haas et al., 2018); inhibiting the SOX9/CA9 (carbonicanhydrase 9)-mediated oncogenic pathway to enhance TMZ-sensitivity (Xuet al., 2018); and co-delivery of TMZ and siRNA targeting the BCL-2 geneusing a folate-conjugated triblock copolymer (Fa-PEG-PEI-PCL, Fa-PEC) ofpoly(c-caprolactone) (PCL), poly(ethylenimine) (PEI) and poly(ethyleneglycol) (PEG) nanocarrier construct (Peng et al., 2018). All of thetherapies combined with TMZ either are used to augment the effect of TMZand/or effect TMZ-resistant GBM cells.

For the IC₅₀ component of the study, for all cells, OKN-007 had asubstantial effect in reducing the TMZ IC50 concentration, so that theGBM cells all became TMZ-sensitive (or more TMZ-sensitive if theyalready were). There should be a cautionary note regarding assessingTMZ-resistance with the G55 cells. The inventors established that alow-passage G55 cell line is actually TMZ-resistant, whereashigh-passage G55 (>30 passages) may become TMZ-sensitive. For the invivo G55 xenograft data, the inventors used a low-passage G55 cell line(<10 passages), which were a similar passage to those used for theinventors' in vitro studies.

From the varying concentrations of OKN-007, it was concluded that aconcentration of 1 mM OKN was just as effective in reducing cellviabilities, compared to the 2 mM concentration for some cell lines(e.g., LN229, U138, G55), whereas for other cell lines (e.g., U251, T98,LN18) 2 mM OKN was slightly more effective in reducing cell viabilities,compared to the 1 mM concentration.

Protein levels seem to indicate that TMZ elevates HIF-1a in all cells,elevates MGMT in most cells (except LN229), and elevates MPG in only T98and U251 cells. OKN-007 for most cells seems to not affect HIF-1aprotein (compared to in untreated cells), that OKN-007 by itself seemsto not effect protein levels of MGMT (compared to untreated cells;except LN18), and that MPG is still elevated in T98 cells with OKNtreatment alone. Of interest, HIF-1a was induced by TMZ in U251, U138,and T98 cells, and then decreased by OKN-007. Combined TMZ+OKN seems tokeep HIF-1a elevated (except for U138 cells), that MGMT protein levelswere not substantially changed with combined TMZ+OKN treatment, and thatMPG is still elevated in U251 cells with combined TMZ+OKN treatment,whereas other cells are not substantially affected (from either TMZ, OKNor combined TMZ+OKN treatments). Specifically, in U251, T98, and G55cells, the level of MGMT increased due to TMZ, but the treatment ofOKN-007 did not inhibit the expression of MGMT.

At the gene level, for TMZ-treated cells, HIF-1a gene-fold changesincrease in all cells (compared to untreated cells). Combined TMZ+OKNtreatment results in some cells increasing HIF-1a gene-fold changes moreso than TMZ alone (e.g., LN229, LN18, U138 and G55), but overall, theredoes not seem to be any substantial decreasing effect on HIF-1α withcombined treatment. There is noted reduced HIF-1α gene expression due tocombined treatment in T98, but it was not significant. OKN alone seemsto only decrease HIF-1α gene-fold changes in the T98 GBM cells comparedto untreated cells. HIF-1a gene-fold changes in

OKN treatment for U138 and U251 seems to still be increased (compared tountreated cells). Unlike protein levels, there is no reduction in HIF-1αby OKN-007 at the gene level. There was a reduction effect of OKN-007only in T98 cells. HIF-1a gene-fold changes may differ in a more hypoxicenvironment, such as in a tumor (in vivo), compared to the normoxicconditions in cells.

There were only modest increases in MGMT gene-fold changes for TMZ aloneor combined TMZ+OKN in only 3 cell lines (U251, T98, LN18) that haddetectable levels. OKN treatment alone slightly increased MGMT gene-foldchanges in U251 cells, but conversely decreased MGMT gene-fold changesin LN18 and T98 cells (compared to untreated cells). Regarding OKN,these results may indicate that OKN alone may decrease MGMT geneexpression, which may decrease resistant for GBM cells.

MPG gene-fold changes (unlike the protein levels) in TMZ treated cellswere elevated more than 2-fold in 3 cell lines (G55, LN18 and U138).With combined therapy, 4 cell lines (G55, LN229, LN18 and U138) hadelevated MPG gene-fold changes (compared to untreated cells). Ofinterest, OKN treatment alone had continued elevated MPG gene-foldchanges for 2 cell lines (G55 and U138), and some decreases for 2 othercell lines (LN18, T98) (compared to untreated cells). There were nodecreases in MGT by OKN-007 that were observed.

From the RNA-seq data for LN18 GBM cells, interesting upregulated genesare RNF149 (stress sensor gene that amplifies p53 response to arrestcell cycle), IDO-1 (involved in human gliomas), and SLC14a2 (endogenoustransmembrane protein upstream-of-mTORC2 (UT2) negatively regulatesactivation of STAT3). Interesting down-regulated genes include SUMO2(overexpression of SUMO in conditions such as brain ischemia andhypoxia, could increase cell survival whereas the knockdown of SUMOexpression has proven to be toxic to cells; and associated with TGFβ1 inresistant glioma cells (Yoshino et al., 2010), HIST1H1C/H1.2 (knockdownof histone HIST1H1C inhibits high glucose induced inflammation and celltoxicity), and PFN1 (profiling-1 phosphorylation is associated withtumor aggressiveness in human glioma) (Liu et al., 2012).

For RNA-seq data for the LN229 cells, interesting down-regulated genesare EGR1 (associated with shorter disease-free survival in patientswhose tumors are ER positive and

HER2 positive; IGF1R signaling pathway is very relevant in drugresistance), XIST (increased level associated with shorter survival andpoorer prognosis), and PRKDC (prognostic biomarker for chemoresistancein breast cancer patients). Interesting up-regulated genes includeZC3H12A (crucial negative regulator of inflammation), RN7SK (potentialanti-proliferative and tumor-suppressive function), SUN-2, and KLHL21.

From further bioinformatics analysis, FOS has the most robustdown-regulation, which can be observed as an OKN effect across all celllines, and all treatment conditions TMZ, OKN, and TMZ+OKN, as well aswhen the data is separated into each treatment condition or cell lineseparately. FOS has also been observed to be sufficient for decreasingcell viability and sensitizing glioblastoma to DNA damage via radiation,so it is possible that this may be helping TMZ cause DNA damage as well(Liu et al., 2016).

MGMT had non-significant changes in many of the conditions; however, forLN229, the loge fold change was ˜3 with a p-value of ˜0.057.

Other MOA that could be associated with TMZ-resistance and that combinedOKN+TMZ therapy may be affecting include TGF-β1 (Yoshino et al., 2010;Wang et al., 2017), and also possibly Akt (Fan et al., 2014), andmacrophages (Zeng et al., 2017). In the inventors' microarray assessmentof F98 rat gliomas treated with OKN, TGF-β1 played a major role in theMOA for OKN efficacy, by down-regulating 57 genes that are commonlylinked to TGF-β1. Perhaps in combined OKN+TMZ therapy, OKN is affectingTGF-β1 and eliciting a response on TMZ-resistant cells.

From microarray analysis of OKN-007-treated vs. untreated rat F98 gliomaRNA, it was established that downregulated genes mainly included membersof integrin and collagen families, which are enriched in theextracellular matrix1 and cell adhesion. Cell viability of tumor tissuestreated with OKN-007 was decreased, whereas cell death was increased viathe downregulation of ANGPT2, DLL4, HPX, IGF1 and TGFB1 genes.“Angiogenesis” was also decreased in OKN-007-treated samples. Severalimmune response genes were notably downregulated, with LBP(lipopolysaccharide binding protein) being the most downregulated.

TGFB1, the last exon of which was downregulated, was the masterregulator of 57 genes. It participates in nearly all processes mentionedabove, and as such may be considered as the main upstream regulatordownregulated by OKN-007.

In addition to integrins and collagens there's notable group ofextracellular matrix glycoproteins, such as lumican (LUM), fibrillin1and 5 (FBN1), laminin (LAMA2). All of them are downregulated, however,matrix metallopeptidase 3 (MMP3), an enzyme degrading all aforementionedproteins, ADAMTS9 and PRSS2, other peptidases, are also downregulated.CD248, expressed in vascular endothelial cells of malignant tumors butnot in normal blood vessels, is downregulated. That suggestsextracellular matrix maintenance and remodeling is active in untreatedglioma samples, and is normalized by OKN-007 treatment.

TGFB1 and a member of TGF-beta proteins LTBP2 (latent transforminggrowth factor beta binding protein) regulating cell adhesion andmigration among other functions, are also downregulated. Similarly,other cell adhesion-related molecules, such as POSTN (periostin) and LUM(lumican) are downregulated. F11r (F11 receptor), an immunoglobulinsuperfamily gene member, is an important regulator of cell adhesion,cell-cell interaction, and formation of tight junction, —is alsodownregulated. Collectively, it is suggestive that cell adhesion isdownregulated.

LBP (lipopolysaccharide binding protein) is the strongest downregulatedimmune-related gene, and majority of its exons are downregulated. Thisgene is involved in the acute-phase immunologic response togram-negative 8 bacterial infections. DMBT1 (deleted in malignant braintumors 1) play a role in the interaction of tumor cells and the immunesystem. Cytokine receptor IL1R1, oncostatin M receptor (OSMR), whichheterosimerizes with IL6 and IL31AR, interferon (alpha, beta and omega)receptor 1 (IFNARl), and F11 receptors are all downregulated.

Overall, processes overrepresented by downregulated genes appear to bebrought back to a normal state, in contrast with untreated gliomasamples, where they are aberrantly regulated. For example, thesingle-exon CD248 gene is highly expressed in malignant tumors (and inthe untreated glioma samples from this study). CD248 was downregulatedin OKN-007 treated samples, suggesting a reversal of the tumor cellsback to a normal state.

As gliomas are highly invasive tumors, the inventors also usedmicrofluidic chambers to see if OKN-007 not only had a role ininhibiting cell proliferation, angiogenesis and increasing apoptosis(Towner et al., 2013), but also has a role in inhibiting glioma cellmigration/invasion (Szopa et al., 2017). The inventors showed thatOKN-007 significantly decreased migration velocity. Glioma cell invasionis dependent on the interaction between the glioma cells andextracellular matrix components such as fibronectin, collagen IV,tenascin-C and fibronectin that stimulate different downstream migrationpathways (Demuth and Berens, 2004).

As TGF-β1 plays a major role in TMZ-resistance (Yoshino et al., 2010;Wang et al., 2017), and OKN-007 may actually be affecting TMZ-resistanceby targeting TGF-β1. Additional supportive data in the LN18 RNA-seq datashows that SUMO2, which is associated with TGF-β1 TMZ-resistance, isalso knocked down when OKN is combined with TMZ (compared to TMZ alone).

Based on the in vitro combined OKN-007+TMZ treatment studies, for futurestudies, the following biomarkers, HIF-1α, TGFβ1, c-FOS, PFN-1, SUMO2,all need to be further pursued as potential key molecular componentsthat may be involved in the role that OKN-007 may play insynergistically affecting TMZ-resistance, when combined with TMZ.

It should be noted that in previous studies for U251 and U87 cell lines,that gene expression profiles generated from tissue culture weresignificantly different from those generated from a subcutaneous (s.c.)implanted tumor, which was significantly different from those grownintracerebrally (i.c.). The disparity between the i.c gene expressionprofiles and those generated from s.c. xenografts suggests that whereasan in vivo growth environment modulates gene expression, orthotopicgrowth conditions induce a different set of modifications (Camphausen etal., 2005). This may imply the importance of using an appropriate modelto correctly represent the tumor microenvironment (TME).

In conclusion, OKN-007 is an interesting anti-glioma agent, which notonly can be effective on its' own, by targeting the tumorigenic TGFβ1pathway via downregulation of key genes associated with theextracellular matrix and cell invasion, but also can elicit an effect onTMZ-resistance GBM cells/tissues. The MOA associated with combinedOKN-007+TMZ therapy does not seem to occur via either HIF-1α, MGMT orMPG. OKN-007 itself did decrease some of these genes and proteins forsome of the GBM cells, but the majority of GBM cells were either notaffected or had elevated levels, with both OKN-007 or combined treatmentgroups. RNA-seq analysis provided some insights to other possible MOAsregarding how OKN-007 may play a role on TMZ-resistance when combinedwith TMZ, and these will need to be further pursued. Combining OKN-007with TMZ seems like a promising therapeutic strategy that may preventTMZ-resistant GBM cells from proliferating, and possibly extending theeffect of TMZ.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method of treating atemozolomide-resistant glioma in a subject comprising administering tosaid subject a dose of 2,4-disulfonyl phenyl tert-butyl nitronc(2,4-ds-PBN) and temozolomide effective to inhibit the vascularization,growth or spread of said glioma.
 2. The method of claim 1, whereinadministration is through a route requiring subsequent passage of2,4-ds-PBN across the blood brain barrier.
 3. The method of claim 1,wherein the subject has a recurrent or metastatic glioma.
 4. The methodof claim 1, wherein the subject has previously failed one or moreanti-glioma therapies.
 5. The method of claim 1, wherein the effectivedose of 2,4-ds-PBN is from about 5 to about 150 mg/kg body weight perday.
 6. The method of claim 1, further comprising a secondaryanti-glioma therapy.
 7. The method of claim 1, wherein said glioma is anastrocytoma, an oligodendroglioma, or a glioblastoma multiforme, or aTGF-β1-, MGMT- and/or APGN-expressing form of any of the foregoing. 8.The method of claim 1, further comprising assessing therapeutic efficacyby measuring the expression of liposaccharide binding protein prior toand after a treatment with 2,4-ds-PBN.
 9. The method of claim 2, whereinthe route is enteral, intravenous, or intra-arterial.
 10. The method ofclaim 2, wherein enteral administration is through dietarysupplementation of a food component.
 11. The method of claim 2, whereinthe enteral administration is in the form of a pill or a liquid.
 12. Themethod of claim 6, wherein the secondary anti-glioma therapy isradiation, surgery, or chemotherapy.
 13. The method of claim 1, whereinan effective dose of temozolomide is lower than the standard monotherapydose of temozolomide.
 14. The method of claim 10, wherein the effectivedose of 2,4-ds-PBN is from about 0.005 w/w % to about 0.1 w/w % of thediet being administered.